Autophagy in Cancer-Regulation by Small Molecules.pdf

Feature ReviewAutophagy in Cancer:Regulation bySmall MoleculesAllison S.Limpert,1,3Lester J.Lambert,1,3Nicole A.Bakas,1Nicole Bata,1Sonja N.Brun,2Reuben J.Shaw,2and Nicholas D.P.Cosford1,*During times of stress,autophagy is a cellular process that enables cells to reclaimdamaged components by a controlled recycling pathway.This mechanism forcellular catabolism is dysregulated in cancer,with evidence indicating that cancercells rely on autophagy in the hypoxic and nutrient-poor microenvironment of solidtumors.Mounting evidence suggests that autophagy has a role in the resistance oftumors to standard-of-care(SOC)therapies.Therefore,there is significant interestin the discovery of small molecules that can safely modulate autophagy.In thisreview,we describe recent advances in the identification of new pharmacologicalcompounds that modulate autophagy,with a focus on their mode of action,valueas probe compounds,and validation as potential therapeutics.The Modulation of Autophagy with Small MoleculesRestoring energy balance during periods of varying nutrient availability is a fundamental abilityof eukaryotic cells 1.This evolutionarily conserved catabolic process is called macro-autophagy(herein autophagy;see Glossary).During autophagy,cells recycle macromo-lecules and whole organelles by forming a compartment called an autophagosome,whichsequesters and traffics its contents for controlled fusion with the lysosome(Figure 1).Autophagy can act as either a prodeath or a prosurvival process and is both tissue andmicroenvironment specific 26.Evidence for this contextual complexity comes from bothobservation of the physiological consequences of genetic impairment of specific autophagygenes and the observed phenotypes in targeted knockout experiments 7,8.Furthermore,some autophagy proteins have noncanonical functions in cellular pathways that control otherprocesses 9,10.However,autophagy is often activated in malignant cancers and,as aconsequence,has an important role in tumorigenesis,tumor evasion and resistance 11.Given the importance of these pathological mechanisms,there is intense interest in thedevelopment of specific therapeutic agents that control autophagy 12.The repurposed autophagy inhibitors chloroquine(CQ)and hydroxychloroquine(HCQ)areclinically approved for the treatment of malaria.These compounds deacidify the lysosome,blocking fusion with the autophagosome.HCQ synergizes with SOC therapeutic agents,butits anticancereffects arecomingledwithmechanismsindependent oftheireffectonautophagy3.A series of small clinical trials combining high doses of HCQ with SOC have been conducted,butthese afforded only modest inhibition of autophagy in patients and yielded promising but incon-sistent results13,14.Significantlimitations in these trials includethe lack of potencyin modulatingautophagy and off-target toxicity,which restricts the therapeutic window 15,16.Herein,we review recent progress in the discovery,development,and therapeutic applicationof small molecules that modulate autophagic pathways,with an emphasis on oncology.Thescope is confined to conserved components of macroautophagic flux,although xenophagyHighlightsAutophagy maintains cellular homeos-tasis during periods of stress.Dysregulation of autophagy has a cru-cial role in cancer and tumor growth.There are several control points in theautophagic pathway that can be tar-geted to modulate the process.Small molecules that modulate autop-hagy by way of inhibition or activationof the autophagic pathway havepotential in oncology.1NCI Designated Cancer Center,Sanford-Burnham Prebys MedicalDiscovery Institute,La Jolla,CA,USA2Department of Molecular and CellBiology,The Salk Institute forBiological Studies,San Diego,LaJolla,CA,USA3These authors contributed equally*Correspondence:ncosfordsbpdiscovery.org(N.D.P.Cosford).Trends in Pharmacological Sciences,December 2018,Vol.39,No.12 https:/doi.org/10.1016/j.tips.2018.10.004 1021 2018 Elsevier Ltd.All rights reserved.and mitophagy have distinct macromolecular components and are also the subject of phar-macological research.Other recent reviews have focused on biological and clinical studiesaround autophagy,whereas this review highlights recent advances in early drug discovery3,1724.Target:ULK1/2 InhibitionAmong the critical factors that regulate the yeast autophagy preinitiation complex is the apicalserine/threonine kinase,ATG1 25.There are five mammalian homologs of ATG1,unc-51-likekinases(ULK)14 and serine/threonine kinase 36(STK36).Of these,only ULK1/2 have beenidentified to be integral to the signaling that drives autophagy 26.While most research to datehas focused on the role of ULK1,including the discovery of small-molecule ligands of ULK1,ULK1 and ULK2 are functional homologs and modulation of both kinases may be critical forautophagy regulation 27.Similar to ATG1,ULK1/2 are the recipients of both negative andpositive phosphorylation by the metabolic sensor proteins mammalian target of rapamycinkinase(mTOR)and 50-AMP-activated protein kinase(AMPK)2831.During nutrientdeprivation stress,mTOR is inactivated while AMPK becomes active(Figure 1),resulting inthe activation of ULK1/2 bound in a complex comprising autophagy-related protein 13(ATG13),ATG101,and FIP200 32.The activated state of ULK1/2 kinase promotes thephosphorylation of several autophagy initiating targets,including Beclin-1 and vacuolarprotein sorting 34(VPS34).From a functional standpoint,the ULK1/2-dependent phosphorylation of components in theinitiation complex is a key regulatory step in the nascent formation of the isolation membraneand/or phagophore and,therefore,is a target of interest for drug discovery.ULK1/2 Inhibitor:SBI-0206965To discover small molecules that can inhibit autophagy at the point of initiation,Egan et al.usedtarget-based reverse pharmacology to develop a small-molecule inhibitor specific for ULK1/233.Starting with a focal adhesion kinase(FAK)inhibitor that also inhibited ULK1,analogswere tested using ULK1 in vitro kinase assays before progressing to cell-based assays toanalyze the phosphorylation state of a consensus sequence in VPS34.This medicinal chem-istry strategy based on structureactivity relationship(SAR)data enabled the development ofthe potent ULK1/2 inhibitor,SBI-0206965(Table 1).In kinetic inhibition assays,this compounddisplayed nanomolar half maximal inhibitory concentration(IC50)values for both ULK1 andits homolog ULK2.Assays to test for target engagement in cells determined that SBI-0206965 inhibits thephosphorylation of VPS34 and Beclin-1,while also eliminating the gel mobility shifts ofATG13 in a manner consistent with a reduction in the ULK1/2 phosphorylation of theseproteins.SBI-0206965 promoted apoptosis in tumor cells dependent on autophagy andreduced autophagy in cancer cell lines following mTOR inhibition.Importantly,when combinedwith mTOR inhibitors,SBI-0206965 induced robust cell death in apoptotic assays.The utility of the chemical probe SBI-0206965 was explored by a separate team of researchers34.Tang et al.demonstrated that non-small cell lung cancer(NSCLC)cell lines have elevatedlevels of ULK1 and upregulated autophagy,which are linked to poor patient outcome.Theyshowed that downregulation of ULK1 expression in NSCLC cells reduced cellular viability andincreased sensitivity to cisplatin,a common NSCLC treatment.They also demonstrated thatcisplatin treatment itself upregulated ULK1 expression in NSCLC cells and that short hairpinRNA(shRNA)knock down of ULK1 increased cisplatin-induced cell death 34.Finally,SBI-Glossary50-AMP-activated protein kinase(AMPK):an enzyme that has acrucial role in regulating ATPproduction and maintaining cellularhomeostasis.Autophagic flux:a measure ofautophagic degradation activity asdetermined by a definedmethodology.Autophagosome:key componentof autophagy;comprises double-membraned vesicles that encloseand isolate cellular material to bedegraded by autophagy.Autophagy:the degradation ofintracellular proteins and damagedorganelles to maintain cellularhomeostasis.Autophagy-related protein 3(ATG3):an ubiquitin-conjugatingenzyme E2 homolog that mediatesthe conjugation of LC3-I tophosphatidylethanolamine after LC3activation by ATG7.Autophagy-related protein 4B(ATG4B):a cysteine peptidaseenzyme,homolog of ATG4,involvedin the cleavage of terminal residuesof proLC3,which activates LC3-I toallow phospholipid conjugation andpromote autophagosome formation.It also cleavesphosphatidylethanolamine from LC3-II.Autophagy-related protein 7(ATG7):an ubiquitin-activatingenzyme E1 homolog that binds toand activates LC3 for conjugation.Autophagy-related protein 13(ATG13):an autophagy proteintarget of the mTOR signalingpathway that regulatesautophagosome formation throughphosphorylation.Beclin-1:an important PI3K proteinthat interacts with BCL-2 or Pl3Kand has a crucial role in theactivation and regulation ofautophagy and apoptosis.FIP200:a component of the ULK1/2complex that regulates early-stageinduction of autophagosomeformation.Focal adhesion kinase(FAK):anonreceptor tyrosine kinase that hasa crucial role in adhesion-mediatedsignaling in cells and is important forautophagy.1022 Trends in Pharmacological Sciences,December 2018,Vol.39,No.12Half maximal inhibitoryconcentration(IC50):theconcentration of an inhibitor in whicha response is reduced by half.Mammalian STE20-like proteinkinase 4(MST4):a serine/threonineprotein kinase that functions tophosphorylate ATG4B and initiate itsactivity.Mammalian target of rapamycin(mTOR):a serine/threonine proteinkinase of the PIKK kinase family thatregulates various cellular processes,including metabolism,cell growth,and autophagy.Microtubule-associated proteins1A/1 B light chain 3B(LC3):anubiquitin-like protein(LC3-I)thatconjugates to the phospholipidphosphatidylethanolamine to formLC3-II and is involved in theformation of autophagosomes.Phosphoinositide 3-kinase(PI3K):a class of signal transducer enzymewithin cells that regulates cellularfunction by phosphorylation ofphosphatidylinositol(PI)-related lipids.p62:a protein kinase C-interactingprotein that is involved in thesignaling and activation of the mTORpathway;degraded by autophagy.Palmitoyl-protein thioesterase 1(PPT1):a lysosomal thioesterasethat cleaves acyl thioester-modifiedproteins in the lysosome.Phosphoinositide-dependent-kinase-1(PDPK1):a serine/threonine kinase which acts as amaster kinase,phosphorylating andactivating a subgroup of the AGCkinase superfamily.Regioisomers:a type of structuralisomer in which two molecules withthe same molecular formula containa functional group or substituent withvarying bonding arrangements.TANK-binding kinase 1(TBK1):aserine/threonine kinase involved inthe innate immune response system.Unc-51-like autophagy activatingkinase 1/2(ULK1/2):two similarserine/threonine kinase homologs ofATG1,which are crucial forautophagy signaling.Vacuolar protein sorting protein34(VPS34):a class III PI3K proteinthat functions to phosphorylatephosphatidylinositol to generatephosphatidylinositol(3)-phosphate(PI3P)necessary for autophagosomeformation.0206965 was shown to destabilize the prosurvival proteins BCL2 and BCLXL,suggesting thatapoptotic pathway activation is a significant factor in the activity of SBI-0206965 and that ULK1is a promising therapeutic target for NSCLC treatment 34.ULK1/2 Inhibitors:MRT67307 and MRT68921Petherick et al.performed an in vitro screen of known kinase inhibitors against ULK1 and ULK235.This led to the discovery of MRT67307 and MRT68921,previously identified as inhibitorsof TANK binding protein 1(TBK1,a component of the innate immune response system),thatexhibited IC50values against both ULK1 and ULK2 in the mid to low nanomolar range(Table 1).Following amino acid starvation to induce ULK1 activity,treatment of mouse embryonicfibroblasts(MEFs)with either MRT67307 or MRT68921 reduced serine 318 phosphorylationon ATG13,an established phosphorylation site for ULK1 36.Treatment with either MRT67307or MRT68921 reduced the processing of microtubule-associated protein 1A/1B-lightchain(LC3)-I to the lipidated LC3-II phosphatidylethanolamine isoform that is associated withactive autophagy(Figure 1)37.Unsurprisingly,in vitro profiling indicated that MRT67307 andMRT68921 display significant off-target effects at TBK1 and AMPK-related kinases.Todetermine whether the reduction in autophagic flux was due to engagement with thesekinases,MEFs with genetic deletions in either TBK1 or liver kinase B1(LKB1),a regulator ofAMPK,were tested under comparable conditions.In these modified cells,the MRT com-pounds reduced LC3-I processing,suggesting that neither TBK1 nor AMPK are required forcompound-dependent autophagy inhibition.Petherick et al.also showed that MRT68921blocked autophagic flux in wild-type MEFs but not in a drug-resistant mutant form of ULK1(M92T),suggesting that MRT68921 blocks ATG through inhibition of ULK1 35.This workexemplifies the context dependence of autophagic modulation,since the off-target inhibition ofTBK1 would be expected to have a significant effect on the closely related process ofxenophagy.ULK1/2 Inhibitors:Compounds 1/3To obtain structural information on the kinase domain of ULK1,Lazarus et al.performed an invitro screen for small molecules that could stabilize the ULK1 protein 38,39.This process ledto the pyrazolylquinazoline derivative Compound 1(as referenced in 39),which both stabilizesand inhibits ULK1(Table 1).A crystal structure of ULK1 was solved with Compound 1 bound inthe ATP-binding site of the active conformation of the kinase.Although equipped with thisstructural information,chemistry optimization around Compound 1 did not provide an analogselective enough for ULK1 to merit use in cells 38.Lazarus et al.next explored pyrimidine derivatives identified in the original kinase screen 39.The compound BX-795(Table 1),a phosphoinositide-dependent-kinase-1(PDPK1)inhib-itor,was the inspiration for chemistry focused on improving selectivity for ULK1,which led to theidentification of Compound 3(as referenced in 39)(Table 1)39.Compound 3 was co-crystalized with ULK1 to obtain a high-resolution crystal structure,which confirmed binding tothe ATP-binding site.In cells treated with Compound 3,a reduction in LC3-I processing wassuggestive of autophagy inhibition.This work is significant because it is the first report of high-resolution crystal structures of inhibitors bound to ULK1.ULK1/2 Inhibitors:SR-17398 and SR-20295Using the ULK1 crystal structure from the work of Lazarus et al.38,Wood et al.initiated an insilico screen for inhibitors that prioritized hydrogen-bonding interactions in the hinge region ofthe ATP-binding pocket 40.The in vitro phosphorylation of ATG13 was used to test predictedhits resulting in the identification of indazole SR-17398(Table 1)41.Molecular modelingTrends in Pharmacological Sciences,December 2018,Vol.39,No.12 1023guided the synthesis of analogs,with high potency achieved with SR-20295,a nanomolar-range ULK1 inh