Defining the Anti-Cancer Activity of Tricarbonyl Rhenium Complexes: Induction of G2/M Cell Cycle Arrest and Blockade of Aurora-A Kinase Phosphorylation.

A Journal of Accepted Article Title:Defining the Anti-Cancer Activity of Tricarbonyl Rhenium complexes: Induction of G2/M cell cycle arrest and Blockade of Aurora-A Kinase Phosphorylation Authors:Peter V. Simpson, Ilaria Casari, Silvano Paternoster, Brian W. Skelton, Marco Falasca, and Massimiliano Massi This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article. To be cited as: Chem. Eur. J. 10.1002/chem.201701208 Link to VoR: http://dx.doi.org/10.1002/chem.201701208 Supported by Chemistry - A European Journal 10.1002/chem.201701208 Defining' the' Anti+Cancer' Activity' of' Tricarbonyl' Rhenium' complexes:' Induction' of' G2/M' cell' cycle' arrest' and' Blockade' of' Aurora+A' Kinase' Phosphorylation' ' % Peter% V.% Simpson,* Ilaria% Casari,% Silvano% Paternoster,% Brian% W.% Skelton,% Marco% Falasca*%and%Massimiliano%Massi*% % Dr.%Peter%V.%Simpson,%Assoc.%Prof.%Massimiliano%Massi% Curtin% Institute% of% Functional% Molecules% and% Interfaces% A% Department% of% Chemistry,% Curtin% University,% Kent% Street,% Bentley% 6102% WA,% Australia.% EAmail:% peter.simpson@curtin.edu.au,% m.massi@curtin.edu.au.% % Prof.%Brian%W.%Skelton% School% of% Chemistry% and% Biochemistry,% University% of% Western% Australia,% Crawley% 6009% WA,% Australia.% % Mrs%Ilaria%Casari,%Mr%Silvano%Paternoster,%Prof.%Marco%Falasca% Metabolic%Signalling%Group,%School%of%Biomedical%Sciences,%Curtin%Health%Innovation%Research% Institute,% Curtin% University,% Perth,% Western% Australia% 6102,% Australia.% EAmail:% Marco.Falasca@curtin.edu.au.% % Abstract' Rhenium% and% ruthenium% complexes% containing% NAheterocylic% carbene% (NHC)% ligands% and% conjugated% to% indomethacin% were% prepared.% The% anticancer% properties% were% probed% against% pancreatic%cell%lines,%revealing%a%remarkable%activity%of%the%rhenium%fragment%as%anticancer%agent.% The%ruthenium%complexes%were%found%to%be%inactive%against%the%same%pancreatic%cancer%cell%lines,% either%alone%or%in%conjugation%with%indomethacin.%An%in%depth%biological%study%revealed%the%origin% of%the%anticancer%properties%of%the%rhenium%tricarbonyl%fragment,%of%which%a%complete%elucidation% had%yet%to%be%achieved.%It%was%found%that%the%rhenium%complexes%induce%cell%cycle%arrest%at%the% G /M%phase%by%inhibiting%the%phosphorylation%of%AuroraAA%kinase.%A%preliminary%study%on%the% 2 structureAactivity%relationship%on%a%large%family%of%these%complexes%revealed%that%the%anticancer% properties%are%mainly%associated%with%the%lability%of%the%ancillary%ligand,%with%inert%complexes% showing%limited%to%no%anticancer%properties.% ! 1! 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Chemistry - A European Journal 10.1002/chem.201701208 Cancer%is%one%of%the%leading%causes%of%death%and,%as%such,%the%drive%to%discover%new%antiAcancer% drugs%is%an%expanding%area%of%research.%One%class%of%frontline%antiAcancer%agents%are%the%platinumA based%drugs%cisplatin,%carboplatin,%and%oxaliplatin,%which%are%extremely%effective%at%eradicating% cancer% cells,% but% suffer% from% severe% sideAeffects% and% poor% activity% against% platinumAresistant% cancers.% Other% organometallic% and% inorganic% complexes% have% been% studied% extensively% as% an% alternative%to%organic%based%drugs.1A4%In%this%context,%complexes%of%gold,%platinum,%and%ruthenium% have%attracted%considerable%attention%and%have%been%shown%to%be%particularly%effective,%often%by% targeting% mitochondria% or% DNA.5%9% Organometallic% complexes% of% rhenium% that% contain% the% chemically%robust%Re(CO) %fragment%have%recently%shown%potential%as%cytotoxic%agents,%but%they% 3 have%been%far%less%studied.%Furthermore,%their%mechanism%of%action%has%not%been%established%in% details%yet,%which%hinders%the%systematic%design%and%investigation%of%targeted%rhenium%complexes% as% anticancer% agents.% All% the% reported% cytotoxic% rhenium% complexes% contain% bidentate% or% tridentate%ligands%of%nitrogen,%oxygen,%and%phosphorus%donors,%a%chemical%design%that%seems%to%be% predominantly%inspired%by%the%use%of%these%species%as%luminescent%cellular%markers.10%Other%rare% examples%include%rhenium%complexes%bearing%cyclopentadienyl%ligands.11% We%and%others%have%been%recently%interested%in%the%investigation%of%the%photochemical%properties% of% rhenium% complexes% bound% to% NAheterocyclic% carbene% (NHC)% ligands.12A18% Surprisingly,% this% relatively%new%class%of%complexes%has%never%been%assessed%in%a%biological%context,%either%as% luminescent%cellular%markers%or%anticancer%agents.%We%therefore%endeavored%to%evaluate%rhenium% NHC%species%against%several%pancreatic%cell%lines%(one%of%the%most%aggressive%cancers%with%a%fiveA year%survival%rate%around%5%)%in%direct%comparison%with%ruthenium%arene%complexes,%whose% structural%motif%has%shown%anticancer%properties%in%previous%investigations.19%For%this%scope,%we% decided%to%study%the%complexes%alone%or%conjugated%to%the%nonsteroidal%antiAinflammatory%drug% (NSAID)% indomethacin.% This% chemical% design% was% inspired% by% the% fact% that% molecular% units% comprising%a%metal%fragment%and%NSAID%have%been%previously%reported%to%enhance%anticancer% properties.20%26% Remarkably,% it% was% found% that% the% rhenium% complexes% were% active% against% pancreatic% cell% lines,% while% the% ruthenium% complexes% did% not% show% any% significant% activity.% Furthermore,%conjugation%to%indomethacin%did%not%seem%to%enhance%the%anticancer%activity,%which% was%traced%back%exclusively%to%the%presence%of%the%Re(CO) %fragment.%With%this%study,%for%the%first% 3 time,% it% was% possible% to% elucidate% that% the% anticancer% properties% of% the% rhenium% tricarbonyl% fragment% originate% from% the% lability% of% the% monodentate% ancillary% ligand% coordinated% to% the% ! 2! 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Chemistry - A European Journal 10.1002/chem.201701208 rhenium% center% by% inducing% G2/M% cell% cycle% arrest% and% blockade% of% AuroraAA% Kinase% phosphorylation.% % Cl O Cl N O N N Br N Br N N N N N Re CO Re CO MeO X N N OC Re Br MeO H N OC CO OC CO 3 (X = O) O OC CO 4b 9 O 4a (X = NH) Cl O O Cl Ru PF 6 N X N Cl Ru PF 6 N MeO N N N MeO OH N N O 7.PF (X = O) O Cl 8.PF 6 (X = NH) 10.PF 6 indomethacin 6 % Figure'1.%Structures%of%rhenium%and%ruthenium%NHC%complexes.% % A% detailed% description% of% the% synthesis% of% the% complexes% shown% in% Figure% 1% is% given% in% the% Supporting%Information.%Briefly,%indomethacinAfunctionalized%azolium%salts%1.Br%and%2.BPh %were% 4 prepared%and%then%reacted%with%Re(CO) Br%to%afford%complexes%3,%4a,%4b.%To%the%best%of%our% 5 knowledge,% this% is% the% first% example% of% an% abnormal% carbene% complex% of% rhenium.% The% spectroscopic% evidence% for% complex% 4b% containing% an% abnormal% carbene% is% discussed% in% the% Supporting%Information.%The%XAray%structure%of%complex%3%can%be%seen%in%Figure%2,%with%additional% data%found%in%the%Supporting%Information.%Reaction%of%1.Br%and%2.BPh %with%Ag O%gave%complexes% 4 2 5%and%6,%which%were%treated%with%[Ru(cymene)Cl] %(cymene%=%1AmethylA4A(propanA2Ayl)benzene)%to% 2 afford%complexes%7.PF and%8.PF .%% 6% 6 % ! 3! 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Chemistry - A European Journal 10.1002/chem.201701208 % Figure'2.%XARay%structure%of%complex%3%with%anion%and%disordered%components%omitted%and% displacement%ellipsoids%at%the%50%%probability%level.% % The%biological%activity%of%the%rhenium%and%ruthenium%complexes%were%tested%on%a%panel%of%human% pancreatic%cancer%cell%lines%comprising%HPAFAII,%ASPC1,%and%CFPAC.%To%understand%if%the%metalA indomethacin%conjugates%possessed%dual%activity,%complexes%9%and%10.PF %as%well%as%indomethacin% 6 alone% were% tested% against% the% same% cells.% Strikingly,% complex% 4b% and% indomethacin% were% ineffective%at%concentrations%up%to%10%µM%(Figure%S11)%while%the%ruthenium%complexes%were% ineffective%up%to%75%–%100%µM%(Figure%S12).%Contrarily,%rhenium%complexes%3,%4a,%and%9%at%10%µM% showed%almost%complete%inhibition.%To%determine%the%effect%of%the%rhenium%compounds%on%cell% growth,% these% cell% lines% were% treated% with% increasing% concentrations% of% specific% rhenium% compounds%(Figure%S13).%We%observed%that%treatment%with%each%inhibitor%significantly%and%doseA dependently%reduced%the%number%of%cells,%as%assessed%by%cell%counting%after%72%hour%incubation%in% complete%media%containing%10%%FBS,%with%IC %values%for%3,%4a,%9,%and%carboplatin%as%control%shown% 50 in%Table%1.%The%IC %values%indicate%that%the%rhenium%compounds%are%remarkably%active%given%the% 50 robust%nature%of%these%particular%pancreatic%cancer%cell%lines,%with%similar%or%slightly%higher%activity% than% carboplatin.% Importantly,% while% the% rhenium% compounds% almost% completely% inhibit% all% pancreatic%cancer%cell%lines%at%10%µM,%around%20%%of%all%the%pancreatic%cancer%cells%in%each%line% are%resistant%to%carboplatin%up%to%50%µM. Furthermore,%since%compound%9%was%as%active,%or%more% ! 4! This article is protected by copyright. All rights reserved. Chemistry - A European Journal 10.1002/chem.201701208 active,%than%the%indomethacin%conjugates%3%and%4a,%we%can%confidently%conclude%that%it%is%the% rhenium%complex%that%is%responsible%for%pancreatic%cancer%cell%inhibition.%To%the%best%of%our% knowledge,%this%is%the%first%example%of%rheniumANHC%complexes%displaying%antiAcancer%activity.% While%there%are%a%number%of%rhenium%complexes%of%diimine%ligands%that%display%moderate%to%high% antiAcancer%activity,%as%recently%reviewed%by%Gasser,%only%two%reports%describe%their%activity%against% pancreatic%cancer,%although%no%explanation%of%the%mechanism%was%given.27,28%Interestingly,%the% active%compounds,%and%in%particular%9,%seem%to%only%be%effective%at%higher%concentrations%(above% 10%µM)%when%tested%on%healthy%human%embryonic%kidney%293T%cells%(HEK293T)%(Figure%S14),% indicating%a%moderate%selectivity%toward%pancreatic%cancer%cells%over%healthy%cells.%To%further% validate%these%results,%pancreatic%cancer%cells%were%treated%with%3,%4a,%and%9%and%cell%viability% assessed%by%the%Alamar%blue%assay.29%The%three%compounds%almost%completely%impair%viability%of% all%pancreatic%cancer%cell%lines%tested,%while%being%less%active%against%HEK293T%(see%Figure%S15A17).% Overall,%these%results%demonstrate%the%great%efficacy%of%the%novel%rhenium%compounds%in%blocking% proliferation/viability%of%different%pancreatic%cancer%cells. % Table'1.%IC %(µM)%values%for%compounds%3,%4a,%and%9%against%pancreatic%cancer%cell%lines.%% 50 Compound% ASPC1% HPAFAII% CFPAC% HEK293T% 3% 7.9%±%1.4% 6.0%±%2.0% 6.0%±%1.8% 11.8%±%2.3% 4a% 9.4%±%3.5% 4.8%±%0.8% 5.4%±%1.4% 8.6%±%0.3% 9' 4.0%±%1.2% 5.6%±%0.6% 5.7%±%2.8% 14.8%±%2.4% Carboplatin% 6.8%±%2.0% 8.7%±%4.3% 7.4%±%1.2% 45a,#30% a%Literature%data.% % The%effect%of%3,%4a,%and%9%on%the%growth%rate%of%the%pancreatic%cancer%cells%was%determined%by%the% Incucyte%Zoom%realAtime%video%imaging%system.%Treatment%with%3,%4a,%and%9%had%a%significant% inhibitory%effect%on%the%growth%of%pancreatic%cancer%cells%in%a%dose%and%time%dependent%manner% compared%to%the%vehicleAtreated%cells.%Representative%video%of%ASPC1%control%cells%and%treated% with%10%µM%9%are%shown%(Suppl.%video).%Moreover,%quantification%of%viable%cells%after%72%hours%of% treatment%showed%that%3,%4a,%and%9%significantly%decreased%the%percentage%of%viable%pancreatic% cancer%cells.%When%pancreatic%cancer%cells%nuclei%were%probed%for%active%caspase%3%or%7%(#4440% IncuCyte),% and% cell% membrane% integrity% assessed% via% Red% CytoTox% permeabilization% (#4632% ! 5! 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Chemistry - A European Journal 10.1002/chem.201701208 Incucyte),%no%major%differences%were%observed%in%cells%treated%with%3%and%9%compared%to%the% vehicle% control% (Figure% S18% and% S19).% In% contrast,% compound% 4a% induced% cell% apoptosis% as% confirmed%using%a%different%assay%(CaspaseAGlo®%3/7%Assay%System).%Taken%together,%these%results% provide%evidence%that%the%rhenium%compounds%3%and%9%do%not%induce%apoptosis%or%disrupt%the%cell% membrane%in%pancreatic%cancer%cells,%but%act%as%cytostatic%drugs.% Our%next%objective%was%to%further%characterize%the%cell%death%process%upon%3,%4a,%and%9%treatment.% Since%treatment%with%3%and%9%did%not%result%in%significant%increase%in%caspaseA3%activity,%we%wanted% to%assess%the%mechanism%of%action%of%rhenium%compounds.%Microscopic%analysis%of%the%pancreatic% cancer%cells%after%treatment%with%3%and%9%did%not%reveal%morphological%changes%associated%with% apoptosis,% instead% we% observed% the% presence% of% multinucleated% cells.% The% presence% of% multinucleated%cells%suggests%cell%cycle%arrest.%Cell%cycle%analysis%after%3,%4a,%and%9%treatment% revealed%a%significant%decrease%in%the%number%of%cells%at%G %with%high%percentage%of%cells%that%were% 1 arrested%in%G /M%phase%(see%Figure%S20).%AuroraAA%plays%a%crucial%role%in%mitotic%entry%and%G2% 2 checkpoint%control.%Dysregulation%of%AuroraAA%induces%abnormal%G2AM%transition%in%mammalian% cells%leading%to%chromosome%instability%and%eventually%in%the%development%and%progression%of% malignant%tumors.%In%addition,%it%has%been%shown%that%Aurora%A%is%overexpressed%in%different% tumors%including%pancreatic%cancer.%Therefore,%we%tested%whether%the%rhenium%compounds%were% able%to%inhibit%the%phosphorylation%of%AuroraAA%in%pancreatic%cancer%cells.%As%shown%in%Figure%3,%10% µM%of%3,%4a,%and%9%completely%inhibit%the%phosphorylation%of%Aurora%A.%Several%studies%have%shown% amplification%and%overexpression%of%AuroraAA%kinase%gene%(AURKA)%in%several%cancers.%Increasing% evidence%demonstrate%that%AuroraAA%plays%a%key%role%in%regulating%cell%cycle%and%mitosis,%as%well%as% a%number%of%important%oncogenic%signaling%pathways.%Therefore,%there%is%an%increasing%interest%in% developing% novel% AuroraAA% inhibitors,% and% some% have% moved% to% phase% III% clinical% trials% in% lymphomas.%However,%despite%the%increasing%evidence%of%the%role%played%by%AuroraAA%in%many% tumors,%there%has%been%a%little%progress%in%the%clinical%development%of%Aurora%A%inhibitors%in%solid% tumors.%Therefore,%it%is%imperative%to%identify%novel%AuroraAA%inhibitors%to%progress%to%clinical% trials.%To%assess%the%tumorigenicity%of%the%rhenium%compounds%in%vitro%against%a%three%dimensional% tumor%model,%we%performed%a%threeAdimensional%soft%agar%colony%formation%assay.%Consistently,% treatment%of%ASPC1,%HPAFAII%and%CFPAC%with%9%inhibited%anchorageAindependent%growth%as%it% reduced%the%number%of%colonies%assessed%by%soft%agar%assays%(Figure%S21).% ! ! 6! 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Chemistry - A European Journal 10.1002/chem.201701208 % % Figure'3.%Regulation%of%AuroraAA%kinase%activity%by%Rhenium%complexes%compared%to%blank%(0.1%% DMSO)%and%positive%controls%(nocodazole%and%gemcitabine).%Western%blot%analysis%of%activated% phosphoAAuroraAA%(Threonine%288)%in%AsPC1%cells%treated%for%24%hours%with%3,%4a%and%9.%%Samples% were%analyzed%for%(top%to%bottom)%AuroraAA%phosphorylation%(ThrA288),%total%AuroraAA%protein% (AuroraAA)% and% αAActinin.% The% same% blot% used% for% phosphoAAuroraAA% was% stripped% and% then% reprobed% with% AntiAAuroraAA% antibody.% Results% are% representative% of% three% independent% experiments.%% % A%preliminary%structureAactivity%relationship%(SAR)%investigation%was%conducted%by%screening%17% complexes%of%the%rhenium%NHC%family%along%with%the%precursor%ligand%imidazolium%salt%and%the% benchmark%luminescent%rhenium%complex%facA[Re(CO) (phen)Cl]%(phen=1,10Aphenanthroline).%The% 3 study%clearly%demonstrates%that%the%anticancer%activity%originates%from%the%degree%of%lability%of%the% ancillary% ligand,% with% inert% complexes% displaying% limited% to% no% anticancer% activity% (see% Supplementary%Information%and%Figure%S22A24%for%a%more%detailed%discussion).%Consistently,%the% two%most%active%rhenium%carbene%complexes%identified%from%the%SAR%were%found%to%inhibit%the% phosphorylation% of% AuroraAA% in% AsPC1% cells,% while% facA[Re(CO) (phen)Cl],% which% contains% a% 3 significantly%less%labile%chloro%ligand,%was%inactive%(Figure%S25).%% Our%results%reinforce%the%potential%usefulness%of%tricarbonyl%rhenium%complexes%as%potent%antiA cancer% agents% against% extremely% robust% pancreatic% cancer% cells.% Most% importantly,% we% have% demonstrated,%for%the%first%time,%that%this%class%of%complexes%acts%as%cytostatic%drugs%by%inducing%a% cell%cycle%arrest%at%the%G /M%phase%associated%with%inhibition%of%the%phosphorylation%of%AuroraAA% 2 kinase.% For% design% purpose,% our% study% indicates% that% the% anticancer% activity% of% the% rhenium% complexes%originates%from%the%lability%of%the%ancillary%ligand.%These%results%shed%some%light%into%the% mechanism%of%anticancer%activity%of%tricarbonyl%rhenium%complexes,%which%was%previously%largely% ! 7! 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Chemistry - A European Journal 10.1002/chem.201701208 unknown%due%to%a%lack%of%systemic%studies.%This%class%of%complexes%could%have%broad%application%as% antiAcancer%drugs.%It%will%be%interesting%to%investigate%both%in#vitro%and%in#vivo%the%cytotoxic% properties% of% these% complexes% and% to% assess% in# vivo% their% antitumor% activity% alone% or% in% combination%with%other%known%chemotherapeutic%agents.% % Acknowledgements' This%work%is%financially%supported%by%the%Australian%Research%Council%(FT1301000033)%to%MM.% Work%in%the%Falasca%laboratory%is%supported%by%Avner%Pancreatic%Cancer%Foundation.%IC,%SP%and%MF% acknowledge%the%infrastructure%and%staff%support%provided%by%CHIRI,%School%of%Biomedical%Sciences% and%Faculty%of%Health%Sciences,%Curtin%University.%The%authors%acknowledge%the%usage%of%the% facilities%at%CMCA,%University%of%Western%Australia.%IC,%SP%and%MF%acknowledge%the%infrastructure% and%staff%support%provided%by%CHIRI,%School%of%Biomedical%Sciences%and%Faculty%of%Health%Sciences,% Curtin%University.%MF%wishes%to%thank%the%support%of%Avner%Pancreatic%Cancer%Foundation.%SP%is% supported% by% Curtin% International% Postgraduate% Research% Scholarship% (CIPRS)/Health% Sciences% Faculty%International%Research%Scholarship%(HSFIRS)% % (1)! 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