Reversible, Spatial and Temporal Control over Protein Activity Using Light

University of Groningen

Reversible, Spatial and Temporal Control over Protein Activity Using Light

Hoorens, Mark W. H.; Szymanski, Wiktor

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Trends in Biochemical Sciences

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10.1016/j.tibs.2018.05.004

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Hoorens, M. W. H., & Szymanski, W. (2018). Reversible, Spatial and Temporal Control over Protein Activity

Using Light. Trends in Biochemical Sciences, 43(8), 567-575. https://doi.org/10.1016/j.tibs.2018.05.004

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Opinion

Reversible,

Spatial

and

Temporal

Control

over

Protein

Activity

Using

Light

Mark

W.H.

Hoorens

and

Wiktor

Szymanski

*

Inbiomedicalsciences,thefunctionofaproteinofinterestisinvestigatedby alteringitsnetactivityandassessingtheconsequencesforthecellor organ-ism.Tochangetheactivityofaprotein,awidevarietyofchemicalandgenetic toolshavebeendeveloped.Thedrawbackofmostofthesetoolsisthattheydo not allow for reversible, spatial and temporal control. Here, we describe selecteddevelopments in photopharmacology thataim at establishing such controloverproteinactivitythroughbioactivemoleculeswithphoto-controlled potency.Wealsodiscusswhysuchcontrolisdesiredandwhatchallengesstill needtobeovercomeforphotopharmacologytoreachitsmaturityasa chemi-calbiologyresearchtool.

TheLimitationsoftheTraditionalToolstoStudyProteinFunction

Cells,tissues, and organisms are highly complex systems in which several thousands of

proteinsinteractandplayaroleinawidevarietyofprocessessuchasmetabolism,signaling, homeostasis,andcelldivision.Tounderstandthefunctionofaproteinofinterestinbothhealth anddisease,researchersalteritsnetactivityandsubsequentlyobservetheresultingchangesin thebiological system[1–3].Tochangethe proteinactivity, awide varietyof chemical and genetictoolshavebeendeveloped.

Bioactivemoleculesarewidelyusedaschemicaltoolstomodifytheactivityofnativeproteins. Themainadvantageisthattheirsolutionscanconvenientlybeaddedtoacellcultureorinjected intoamodelorganism.Formanyproteins,bioactivemoleculeshavebeendevelopedthatcan activateorinhibittheactivity viaeithercompetitiveorallostericmechanisms.Currently,the Bindingdatabase(www.bindingdb.org)reportsover600000smallmoleculestargetingover 7000proteintargets.However,drawbacksofusingbioactivemoleculesincludethelackof reversibilityandlimitedspatialcontrol:thesolutionsareaddedsystemically,andthereisno easywaytoremovethebioactivemoleculeinacontrolledmanner,onceithasbeenadded. Genetictoolsforproteinactivitymodulation,besidescontrollingtheactivityofnativeproteins, canalsochangetheconcentrationoftheproteinofinterestateitherthetranscriptionlevelorthe translationlevelby(singleordouble)knockout,knockdown,andtheuseofsiRNA[4].However, itisknownformanyproteinsthatknockoutsinmicearelethal[5],whichonlydemonstratesthat theseproteinsarecrucial,withoutelucidatingtheirrole.Decreasingtheactivity canalsobe achievedbymaking specificmutations inthe activesite, bya knockin,which resultsina catalyticallyinactiveproteinthatstillmaintainsitsbindingproperties[6].Increasingthe con-centrationofproteinscanbeachievedthroughoverexpression,resultinginhighernetactivityof theproteinofinterest.Genetictools,whilewidelyapplied,areelaborateinuse.Yet,therapidly growingfieldofclusteredregularlyinterspacedshortpalindromicrepeats/CRISPR-associated protein9(CRISPR/Cas9)mightalloweasiermodification[7].Moreadvancedgenetic

techni-ques are inducible expression systems in which addition of a chemical inducer such as

Highlights

Usinglightinmedicineisincreasingly popular,duetothefactthatlight is orthogonal with biological systems and can be regulated and dosed easily.

Inthe past5 years,manybioactive moleculeswithphoto-controlled activ-ityhavebeendeveloped,andthefield of photopharmacology is rapidly expanding.

The chemical toolbox of photo-switches is growing, with a special interest in red-light-operated photo-switches,sinceredandnear-IRlight showsthehighesttissuepenetration.

1

UniversityMedicalCenterGroningen, DepartmentofRadiology,University ofGroningen,Hanzeplein1,9713GZ Groningen,TheNetherlands

2

CentreforSystemsChemistry, StratinghInstituteforChemistry, FacultyofScienceandEngineering, UniversityofGroningen,Nijenborgh7, 9747AGGroningen,TheNetherlands

*Correspondence:

doxycyclinechangestheactivityofapromotorandtherebytheexpression[8],whichcanbe returnedtoitsoriginallevelbywashingoutofthechemicalinducer.Inconclusion,genetictools aremainlyirreversible,thatis,theconcentrationofknocked-outproteincannotbeconveniently restoredto the naturallevel at a given time. Furthermore,the spatial resolution ofprotein expression modification is limited, meaning that, for example, a protein is knocked out systemicallyandnotinanorgan-ortissue-specificmanner.

WhyIsReversible,SpatialandTemporal Controlover ProteinActivity Important?

Currently, the toolbox to alter protein activity relies mainly on irreversible techniques, as discussedpreviously.Yet,reversibilitycanbeofimportanceinelucidatingthe functionofa proteinofinterest.Fromanexperimentalpointofview,reversibilityservesasastrongcontrol, sincethesamesystem(cell,tissue,organism)canbestudiedoverashortperiodoftimewith andwithoutthealteredproteinactivity.Also,reversibilityofmodulationminimizesthe irrevers-ibledownstreameffects,whichareobservedforeveryalterationofabiologicalsystem and dependonthedurationofalteration.Awell-establishedexampleisdrugaddictioninwhichlong dosageofanactivecompoundresultsinadifferentresponsethantheinitialresponse[9,10]. Anothertypicalexampleishowtumorcellscanacquiredrugresistancebyactivatingalternative pathwaystobypasstheinhibitedpathway[11,12].Compensationeffectsandtheirinfluenceon theobservedbiologicaloutcomecouldbebetterunderstoodwhenthedurationoftheinhibition oractivation is precisely controlled. Altogether, reversibility and temporal control over the modulationwillcontributetoabetterunderstandingofproteinfunctioninabiologicalsystem withminimalizedcompensationeffect.

Alteration ofprotein activityby geneticand chemical toolsis mainlysystemic.However,a proteinofinterestmighthaveaspecificfunctioninanorganortissue.Thesystemicalterationof theactivityofaproteinofinterestprovidesobservationsthatcanbedifficulttotracebacktoa specificlocalfunction.Forexample,forhistonedeacetylase2(HDAC2)itwasshownthatthe expressioninthe dorsolateralprefrontalcortexinschizophreniapatientsisdecreased[13]. SinceHDAC2isexpressedinmanytissues[14],systemicinhibitionofHDAC2inananimal modeldoesnothelptoelucidatethespecificroleofHDAC2inthisbrainregion.However,this limitationcouldbeovercomebylocallyinhibitingHDAC2activity,mimickingthepatientsituation morecloselyandcontributingtoabetterunderstandingoftheroleofHDAC2inspecificbrain regionsandtheirconnectiontootherareas.Suchsite-specificalterationsoftheactivitywillalso have large implications in, for example, proving the site of action of drugs, studying cell signaling,andunderstandingadverseeffectsoftherapeutics.

LightIsanEmergingExternalStimulustoControlProteinActivity

Toachievereversible,spatialandtemporalcontroloverproteinactivity,amodulatorisneeded whoseactivitycanbecontrolledwithanexternalstimulus,suchasphotons.Lightisalreadywidely usedinbiologicalstudies,forexample,inopticalandfluorescencemicroscopy,whichisenabled bytheorthogonalityofphotonstowardlivingsystemsandprocesseswithinthem[15].EvenUV lightis,toalargeextent,toleratedincellcultures,asdemonstratedbytheimagingoftheblue fluorescentprotein[16]andDNA-labelingdye40,6-diamidino-2-phenylindole(DAPI)[17].Yet,itis recommendedtodocontrolexperimentsinwhichthebiologicalsystemissubjectedtoirradiation only,tocheckforanyundesiredeffects.Thekeybenefitofusinglightisthatitiseasilypossibleto regulatewhen,where,forhowlong,andwithwhichintensityandwavelengthitisused. Currently,thereareseveraltoolsavailabletouselighttogaincontrolovertheactivityofproteins.

A well-established exampleis optogenetics, where responsive elements from photoactive

proteinsaregeneticallyengineeredintootherproteins,bywhich,forexample,areceptorcan beactivatedwithlightinsteadofachemicalligand[18].Thefieldacknowledgesthedemandof spatialandtemporalcontrolovertheactivityofbiologicalpathways[19].However,expressing engineeredproteinsischallenging.

Achemical approach to acquirephotocontrolisphotocaging. A photocageis a

photores-ponsivechemicalgroupthatusestheenergyofaphotonto breakachemicalbond[20].A photocageisplacedatafunctionalgroupofabioactivemolecule[21]oraminoacidofaprotein

[22]bywhichitlosesitsactivity;uponirradiationthephotocageisremoved,resultinginthe releaseofabiologically activemolecule [23].Theapproachof usingphotocagedbioactive compoundswassuccessfullydemonstratedinvivoinamousemodel[24].Adrawbackisthat thephotochemicalprocessofuncagingisirreversible.

Afullypharmacological,remote,andreversiblecontrolofproteinactivitywithlightisenabled throughtheuseofmolecularphotoswitches,thatis,smallphotoresponsivemoleculesthatupon irradiationchangetheirstructure[25,26](foradetailedexplanation,seeBox1),hencethename

photoswitch.A widely used photoswitchis azobenzene in which the diazobond (N¼N) is

connectedtotwophenylringsthatcanbeonitsoppositesides(trans-azobenzene)oronthe sameside(cis-azobenzene).Thetransisomeristhermodynamicallystableandbecanswitched intothecisisomerbyirradiationwithUVlight(Box1).Thisprocesscanbereversedspontaneously usingheatorthemoleculecanbeswitchedbackusingvisiblelightirradiation.Theprocessof switchingfromtranstocisandbackcanusuallyberepeatedformanycycles[25,27].

The emerging field of photopharmacology utilizes the differences in shape and chemical

properties between photo-isomers of a bioactive moleculethat differ in activity (Figure 1, KeyFigure)andthatcanbeinterconvertedwithlightirradiationand/orspontaneousthermal relaxation[28].Photoswitchessuchasazobenzeneareintroducedintothestructureofthe bioactivemolecule[29].Throughthis,remotecontroloveritsactivity,andthereforetheactivity oftheprotein ofinterest,can beachieved. Photopharmacologymainlyaimsat developing therapeuticsthatareonlyactiveatthetargetandnotinhealthytissue,toeliminateactivityof drugsinhealthy tissueand itsconsequences[30].However,besides this potentialclinical application,bioactivemoleculeswithphotocontrolledactivitycanserveasapowerfultoolin biomedicalresearch.Theseremotelycontrolledbioactivemoleculescansimplybepipettedto acellcultureorinjectedintoamodelorganism;afterwards,bypreciseirradiation,controlover proteinactivityisacquired.Inthefollowing,welookatexamplesfromtheproteinclassesof enzymes,structuralproteins,andreceptorsforwhichphotopharmacologicalcontrolhasbeen establishedeitherinvitroorinvivo.

Photo-controloverEnzymatic Activity

Enzymesaretheworkhorsesofthecellandharbormanyregulatoryfunctionsandprocesses thatareoften dysregulated indisease.To demonstratephotopharmacologicalcontrolover enzymeactivity,thespecificcaseofHDAC2isdiscussedhere.Thisenzymeisamemberofthe histonedeacetylasefamily,whichisinvolvedinepigeneticregulationofgeneexpression[31].In severalcancers,increasedexpressionofHDAC2isobserved,resultingindecreased expres-sionofgeneswithantitumoractivity[14].Therefore,inhibitionofHDAC2hasbeenshowntobe effective in killing tumor cells [32], like, for example, the FDA-approved HDAC2 inhibitor vorinostatforthetreatmentofmetastaticmelanoma[33].

TraditionalgeneticandchemicaltoolboxeshavebeenusedtostudythespecificroleofHDAC2. Unfortunately,HDAC2knockoutmicedieofcardiacmalfunctionthefirstdayafterbirth[32],

demonstratingtheimportance oftheprotein,butnotitsspecificfunction.Todecreasethe

HDAC2 activity pharmacologically, a wide variety of inhibitors have been developed with

selectivity for HDAC2 over other HDACs from the same protein family [33]. Recently, a

photocagedvariantofvorinostatwasdevelopedbywhichspatialandtemporalcontrolover HDAC2activitycanbeachieved[34],howeverirreversibly.

Box1.UnderstandingLight-ControlledDrugs:MolecularStructureandPhotochemistry

Azobenzene(A)isthemost-often-usedmolecularphotoswitchinphotopharmacologyandserveshereasanexampleto introducethebehaviorofmolecularphotoswitches.Azobenzenehastwoisomers:thethermallystabletransisomer (blue)andthethermallyunstablecisisomer(orange).Thesetwoformsdifferinstructure,polarity,solubility,andmany otherfeatures.

Importantly,theirUV-visiblespectraarealsodifferent(B),whichleadstothepossibilityofselectivelyaddressingeachof theformswithlight.Thetransformshowsastrongabsorptionbandatlowwavelengths(denotedasl1;typically,UV

lightof320–370nm),wheretheabsorptionofthecisformislower.Athigherwavelengths(denotedasl2;typically,

visiblelightof420–480nm)thecisformabsorbsmorestronglythanthetransform.Usingl1,itisusuallypossibleto

selectivelyswitchthetransformtothecisform.Withl2,thecisformcanselectivelybeswitchedbacktotrans.

Thefirstoftheseprocessesisdiscussedinmoredetailin(C).Whenlightofl1isapplied,thetransformabsorbsthe

photonandenterstheexcitedstate,fromwhichitcanrelaxtothegroundstateofthecisform.Thekineticsofthis processdependson(i)theprobabilityofabsorbingthephoton,representedbytheextinctioncoefficient ;and(ii)the probabilitythat,onceintheexcitedstate,itwillfalltogroundstatewithisomerization,representedbythetrans-to-cis

isomerizationquantumyieldwt!c

.Whiletheconcentrationofthecisformincreases,italsoabsorbslight,withextinction coefficientof ,andwiththequantumyieldofwc!t

,itcanisomerizebacktotrans.Intime,adynamicequilibriumis establishedbetweenthetwoprocesses.Assumingnegligiblethermalcis–transreisomerizationonthetimescaleofthe experiment,thepositionofthisequilibriumisdescribedbythephoto-stationarystate(PSS),whichissimplythe percentageofcompoundsthatareinthecisstateatequilibriumunderirradiation.

Oncethelightisswitchedoff(D),themolecularphotoswitchreturnstoitsoriginalstate,whichisusually>99%ofthe stabletransform.Thisrecoveryisafirst-orderprocess,andthetimeneededtoisomerizehalfoftheciscompounds backtotransisdescribedashalf-life(t0.5).Thisvaluedependsbothonthestructureofthephotoswitchandonits environment(solvent,temperature,etc.)andcanrangefrommicrosecondstoyears.

(A) (B) (D) (C) Photoswitchable drug PhotoisomerizaƟon UV-visible spectra Trans

Thermally stable Thermally unstableCis

Thermal relaxaƟon

ελ1φt c ελ1φc t ∝ PSS =_[Trans[Cis_{] + [}] _Cis]

[Cis] [Cis] [Cis]/2 [Trans] ελ1 ελ1 Trans Cis Conc. Conc. λ1 λ1 t_0.5 λ1 λ2 λ2 λ2 Wavelength (nm)

Irradiation time withλ1 Time

ε

Toachievethedesiredreversible,spatialandtemporalcontroloverHDAC2activity,ourlab developed HDAC2inhibitors withphoto-controlled activity [35], as shown inFigure 2. For compound1,thecisisomeris39timesmoreactivethanthetransisomer.Thedifferencein cytotoxicactivitybetweentransandciswasalsoobservedinHeLacells,evenshowingalarger differenceincellviabilitythanfortheindividualHDAC2inhibitor.Also,reversibilityandtemporal controlovertheactivityofHDAC2weredemonstrated,overcomingthelimitationsofthecurrent chemicalandgenetictoolbox.

CanBioactive MoleculeswithPhoto-controlledActivityBeDevelopedfor EveryProtein?

Currently,therearehundredsofthousandsofsmallmoleculecompoundsthatcanmodulate the activity of several thousands of target proteins. In contrast, only several dozens of

bioactive molecules with photo-controlled activity have been developed [30]. However,

the numberis rapidlygrowing, andthe listof proteintargetsis expanding.Photo-control overtheactivityofmembersofproteinfamiliessuchasenzymes[36,37],receptors[38–42], transporters [43], and structural proteins [44–47] has been achieved, demonstrating the generalityofthisapproach.Thedesignisusuallybasedonknownproteinmodulatorsthatdo notharborphoto-control.AsshownbytwoexamplesinFigure3,chemicalstructuressimilar

to azobenzene are replaced by an azobenzene photoswitch in a photopharmacological

KeyFigure

The

Principle

of

Photopharmacology,

Explained

with

the

Example

of

a

Photo-regulated

Enzyme

Inhibitor

Ac

Ɵ

vity

[I]

Log[I]

Figure1.(A)Amodelofphotopharmacology.Aninhibitorcontainingaphotoswitchinits‘off’state(blue)hasnostronginteractionswiththetarget;however,inthe‘on’

state(orange),theinhibitorbindsstrongly.Lightofwavelengthl1switchestheinhibitorfromtheoffstatetotheonstate,andlightofwavelengthl2reversesthisprocess.

(B)Dose–responsecurveofabioactivemoleculewithphoto-controlledactivityasshownin(A).Theonstate(orange)ispotentatlowerconcentrationthantheoffstate (blue),anditispossibletoswitchbetweenthosestatesusinglightandthermalrelaxationprocesses.Atacarefullychosenconcentration,[I]opt,theonstatenearlyfully

(A) (B) (D) (C) CombretastaƟn A4 VU0414374 Compound 2trans 2a:IC50 = 50 μM 2b:IC50 = 110 μM Compound 3trans IC50 = 297 nM Compound 3cis IC50 = 1.49 μM Compound 2cis 2a:IC50 = 0.16 μM 2b:IC50 = 0.20 μM 2a: R = Methyl 2b: R = Ethyl 390–430 nm 360–400 nm 420–450 nm heat 500–530 nm heat Control 390 nm Dark Dark Illum385nm Vehicle Nai ve 3 Pa w li Ō s (% of con tr ol) 0 50 100 ns ns **** 1.5 μM 2a

Figure3.ExamplesofBioactiveMoleculeswithPhoto-controlledActivity.(A)Lightcontrolofastructuralprotein:formationofmicrotubule.Basedontubulin

polymerizationinhibitorcombretastatinA4,compounds2aand2bweredesigned.UponirradiationwithUVlight,compound2bbecomes550timesmoreactive,which canbereversedusingvisiblelightirradiation[46].(B)Compound2ainducedthebreakdownoftubulin(green)andfragmentationofthenucleusuponirradiationwith 390nmtotheactivecisisomerand20-hincubation,whileirradiationwithoutinhibitorandthetransisomerofcompound2adonotchangethephysiologyofthecell. Adaptedfrom[45].(C)Lightcontrolofreceptoractivity:metabotropicglutamatereceptor5(mGlu5).BasedonnegativeallostericmodulatorVU0414374,compound3

wasdesigned.UponirradiationwithUVlight,compound3becomes5.1timeslessactive,whichcanbereversedusingvisiblelight[40].(D)Persistentinflammatorypain wasinducedinamousemodel,andafter10daysthenumberofpawliftswasrecorded(naive)andnormalizedtohealthymice(vehicle)withandwithoutirradiationinthe amygdala.Injectionofcompound3resultedinthesamebehaviorinthemouseasinnaivemice;uponirradiationtothecisisomer,thiseffectcouldbeabolished,tothe samelevelasinthevehiclemice.Adaptedfrom[40].

(A) (B)

Log[I] (μM)

HeLa cell viability (%)

Vorinostat Compound 1 trans

IC50 = 21.7 μM Compound 1 cis IC50 = 555 nM 360–410 nm 460–500 nm heat 0 0 -1 1 2 50 100 Trans_Cis

Figure2.Photo-controlovertheActivityofHistoneDeacetylase2(HDAC2).(A)BasedonknownHDAC2inhibitorvorinostat,compound1wasdesigned.

Uponirradiation,compound1switchesfromtranstocisform,becoming39-foldmoreactiveasanHDAC2inhibitor.(B)Dose–responsecurveforcompound1intrans

(blue)andcis(orange)formoncellviabilityofHeLacells.Reproduced,withpermission,from[35].

approach called azologization [48].This approach has been extended toother chemical structureswithlesssimilaritytothestructureofthephotoswitch,guidedbystructure–activity

relationship studies and computational support [40,42,49]. So far, the development of

bioactive molecules with photo-controlled activity is limited by the availability of known modulatorsandtheexistenceinthosemodulatorsofstructuralfeaturesthatcanbereplaced byaphotoswitchwithoutamajorlossinpotency.

The replacement of a fragment of a molecule by a photoswitch has been convincingly

demonstratedbytakingadvantageofthestructuralsimilarityofnaturalcompound combre-tastatin A4 and cis-azobenzene [44,47] (Figure 3A). Combretastatin A4 is an inhibitor of microtubuleformation. Microtubules belong to the family ofstructural proteinsandare an importantcompartmentofthecytoskeleton,playingaroleinmechanicalprocessessuchasthe intracellulartransportofvesiclesandseparationofchromosomesinmitosis[50].Azologization ofcombretastatinA4resultedinaninhibitorwithphoto-controlledactivity(Figure3A),where irradiationoftheinactivetransisomertothecisisomerincreasesthepotencyinHeLacellsin vitrobyanimpressivefactorof550forcompound2b[47].

Reversiblespatialandtemporalcontroloverproteinactivityshowsitsfullpotentialinaninvivo

model.Recently,severalinvivostudiesofphotopharmacologicalagentshavebeenreported, mainlyforneurologicaltargets,suchasrestoringthevisualfunctionoftheblindretina[51],and metabotropicglutamatereceptors[40,52].Animpressiveexampleofaninvivo-tested bioac-tivemolecule with photo-controlled activity was reported by the groupsof Gorostiza and Llebaria,targetingmetabotropicglutamatereceptor5[40,49,53–55],whichisapotentialtarget for the treatment of anxiety, depression, and schizophrenia [56,57]. Inspired by negative

allostericmodulatorVU0414374, compound3 was designed(Figure 3C)and testedinan

invivosystemusinghybridopticandfluidcannulasthatwereimplantedintheamygdalaof persistentinflammatorypainmousemodel.Themousewasinjectedwithcompound3inthe amygdalaintheactivetransconfiguration,resultinginananalgesiceffect.Thispain-relieving effectcouldbeabolishedbyirradiationtotheinactivecisisomer[40].Bythis,photo-control overpaininarodentmodelwas achieved,which opensopportunitiesinstudyingpain,its development,anditstreatment.

TheCurrentLimitationofPhotoswitchableBioactiveMoleculesasa ResearchTool

A challenge inthe development of bioactive molecules withphoto-controlled activity is to acquirelargedifferencesinactivitybetweenthephoto-isomers.AsshowninFigure1B,ata preciselychosenconcentration,[I]opt,oneisomerdoesnotchangetheactivityoftheproteinof interest,while theotherisomer resultsincompleteinhibitionof proteinactivity; hence,the proteincanbeswitchedfullyonandfullyoff.However,thisoptimalsituationoffullyswitchingis rarelyachieved.Forexample,forcompound1,a39-folddifferenceinactivitybetweenthetrans

andcisisomerisnotyetsufficienttoallowforswitchingbetweenfullyactiveHDAC2andfully inhibitedHDAC2 [35].In theoptimizationof photopharmacological agents,everychemical modificationofthebioactivemoleculepotentiallynotonlychangesthebiologicalactivitybut alsothechemicalpropertiesandimportantphotochemicalpropertiessuchastheabsorption maxima,half-lifeofthecisisomer,quantumyield,andthephoto-stationarystates(PSSs).This optimizationprocessischallenging;yet,toreachfullpotentialasaresearchtool,differencesin

theactivitybetweenisomersshouldbeenhanced.

AnotherchallengeisthatmostofthephotopharmacologicalagentsneedUVlightintheregionof 350–400nmtoswitch[30].Suchlighthasalimitedpenetrationdepthofonlyafewmillimetersin

softtissue[58].Thisissufficientforexperimentsinmonolayercellculture,butnotforanimal models,sincemostinnerorganscannotbereachedinanoninvasivemanner.However,redand near-IRlighthasdeeperpenetrationdepthinsofttissue,uptoseveralcentimeters[58].Therefore, red-light-responsivephotoswitchesandphotopharmacologicalagentsareindevelopment[59– 61].Recently,anelegantexamplewaspublishedbytheFeringagroup[62],whereanantibiotic wasdevelopedthatincreaseseighttimesinpotencyuponirradiationwithredlight.

ConcludingRemarksandFutureProspects

Inadditiontothethreeexamplesdescribedhere,formanyotherproteins,bioactivemolecules withphoto-controlledactivity have beendevelopedin recentyears.Besides theirpotential clinicalapplicationsinphotopharmacology,thesearepowerfultoolsforbiomedicalresearch, becauselightisorthogonalwithbiologicalsystems,nogeneticmodificationsarerequired,and

spatialand temporal controlcan be achievedina reversiblemanner. Thebroad rangeof

proteinsthat can be altered by photopharmacology and especially the reversibility of the modificationcanmakeitasuperiortoolcomparedtotheexistingtoolbox.

Morebioactive moleculeswithphoto-controlledactivity will bedeveloped,witha focuson visiblelightswitchingandoptimizationofthedifferenceinactivitybetweenisomers.Inparallel, newphotoswitchesthatcanbeoperatedwithvisiblelightorthathaveenlargeddifferencesin

structurebetweenisomersarebeingdiscovered.Thesedevelopmentswill,moreandmore,

allowphoto-controlledbioactivemoleculesinbiomedicalresearchtocontributetothe under-standingoftheroleofaproteinofinterestinhealthanddisease.

Acknowledgments

ThisOpinionarticlewasfinanciallysupportedbytheNetherlandsOrganizationforScientificResearch(NWO-CW)VIDI grant723.014.001toW.S.

AppendixA SupplementalInformation

Supplementalinformationassociatedwiththisarticlecanbefound,intheonlineversion,athttps://doi.org/10.1016/j.tibs. 2018.05.004.

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OutstandingQuestions

Currently, most bioactive molecules withphoto-controlledactivityhavea difference in activity between the photo-isomers of about 10–50-fold. So,howdowerationallydesignnew compoundswithalargerdifferencein activity?

Eventhoughthereisadifferencein activitybetweenbothphoto-isomers, usually bothcan bindtothe target. Does the bioactive molecule with photo-controlledactivityswitchwhile bound,ordoesitfirsthaveto dissoci-atefromthetargetprotein? Howcaneffectiveandpreferably non-invasiveirradiationofbioactive mole-culeswithphoto-controlledactivitybe achieved in deep organs of model organisms?

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