Subunit specific inhibitors of proteasomes and their potential for immunomodulation

Subunit specific inhibitors of proteasomes and their potential for immunomodulation

Alexei F Kisselev

and Marcus Groettrup

Specializedvariantsoftheconstitutive20Sproteasomeinthe immunesystemliketheimmunoproteasomesandthe thymoproteasomecontainactivesite-bearingsubunitswhich differintheircleavageprioritiesandsubstratebindingpockets.

Theimmunoproteasomeplaysacrucialroleinantigen processingandforthedifferentiationofpro-inflammatory Thelpercellswhichareinvolvedinthepathogenesisof autoimmunity.Selectiveinhibitorsoftheimmunoproteasome andconstitutiveproteasomehaverecentlybeengenerated whichinterferewiththedevelopmentandprogressionof autoimmunediseases.Herewedescribetheseinhibitorsand theirtherapeuticpotentialaspredictedfrompreclinicalmodels.

Addresses

1DepartmentofPharmacology&Toxicology,NorrisCottonCancer Center,GeiselSchoolofMedicineatDartmouth,OneMedicalCenter Drive,Lebanon,NH03756,USA

2DivisionofImmunology,DepartmentofBiology,UniversityofKonstanz, D 78457Konstanz,Germany

3BiotechnologyInstituteThurgau(BITg)attheUniversityofKonstanz, CH 8280Kreuzlingen,Switzerland

Correspondingauthors:Kisselev,AlexeiF (Alexei.F.Kisselev@Dartmouth.EDU)

andGroettrup,Marcus(Marcus.Groettrup@uni konstanz.de)

Introduction

Theubiquitin-proteasomepathwayisthemajorquality- controlpathwayfornewlysynthesizedproteinsinevery eukaryoticcell.The26Sproteasomeisalarge(2.5MDa), multi-subunit,ATP-dependentproteolyticcomplexthat processivelydegradesproteinsintopeptides.Itconsistsof ahollow cylindrical20Sproteolyticcoreandoneor two 19S regulatory particles (RP). The 19S RP recognizes ubiquitylatedsubstrates and preparesthem for proteol- ysis,whichoccursinsidethe20Score.The20Scoresare hollow cylindrical structures comprising two pairs of 14differentpolypeptidesarrangedinfourstackedrings.

Threesubunitsoneachinnerringcarrycatalyticresidues

fortheproteolyticsites(Figure1).Thecatalyticresidues ofthechymotrypsin-likesitesarelocatedonb5subunits and cleave peptide bonds predominantly after hydro- phobic residues. The caspase-like sites are located on theb1 subunitswhichcleavepeptidebondsafteracidic residues. The third pair of sites is located on the b2 subunits. They cleave after basic residues and are referred to as trypsin-like sites. The lymphoid tissues containimmunoproteasomes, in which b5, b2, b1 sub- unitsare replacedwithb5i(LMP7),b2i(MECL1),b1i (LMP2) subunits, respectively (Figure 1). Moreover, epithelialcellsin thecortexof thethymusexpressthy- moproteasomesconsistingoftheactivesitesubunitsb5t, b2iandb1i.Herewedescribetherecentdevelopmentof inhibitors for these tissue specific proteasome variants and howthey canbe exploited for immunomodulation in antigen processing and the therapy of autoimmune diseases.

Themajority ofproteasome inhibitorsinhibit multipleactivesites

The inhibitorsof proteolytic sites of the20S corehave servedasindispensibletoolstoidentifyproteasomesub- stratesandestablishproteasomeinvolvementinavariety of biochemical, immunological, and physiological pro- cesses for two decades (see [1] for review). One of theearliestfindingswiththeseinhibitorswasthattrans- formedcells rapidly undergo apoptosis upon treatment withproteasome inhibitors, while muchhigherconcen- trations are needed to induce death of their non-trans- formed counterparts. These discoveries stimulated developmentof proteasomeinhibitorsas anti-neoplastic agents and, in 2003, bortezomib was approved by the FDAforthetreatmentofmultiplemyeloma.Ittookfew additionalyearstorealizethatexquisitesensitivityofthis particularcancertoproteasomeinhibitorsiscausedbyan exceptionallyhigh load onthe proteasomein myeloma cells, which secrete large amounts of immunoglobulins [2]. In 2012, another proteasome inhibitor, carfilzomib [3], has been approved for the treatment of multiple myeloma.

Because results of site-directed mutagenesis in yeast revealedthatthechymotrypsin-likesitesaremostimport- antinproteindegradation,andbecauseoftheabilityof hydrophobicpeptidestoentercells,thevarioussynthetic proteasomeinhibitorswereoptimizedaccordingtotheir capacitytoblocktheb5sites,whichcleavepreferentially afterhydrophobicresidues[1].Theprimarytargetofall natural product inhibitorsis also thechymotrypsin-like

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-264534

Figure 1

LU-001i ML604440

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Current Opinion in Chemical Biology

Subunits of the constitutive proteasonne (left) and the

immunoproteasome (right) with assignment of their respective inhibitors as mentioned in the text.

site. All compounds targeting chymotrypsin-like sires (e.g., MG-132, bortezomib, epoxomicin, clasro-lactacys- rin-13-lacrone) effectively inhibit protein degradation inside the cells. However, none of these inhibirors can be considered chymotrypsin-like sires selective, and inhi- bition of protein degrada cion occurs at concentrations that co-inhibit either caspase-like or trypsin-like sites [4], raising the possibility that contribution of these two sites

tO protein degradation is larger than previously appreci- ated. This observation, together with the fact that bor- rezomib inhibits the caspase-like activity of the proteasome [4 6], stimulated the interest in the devel- opment of specific inhibirors of the individual active sires, ro be used as rools ro dissect contributions of individual sires ro protein degradation, and ro define their individual roles as targets for anti-neoplastic and ami-inflammatOry agents.

Subunit-specific proteasome inhibitors

The great majority of site-specific proteasome inhibirors consist of an elecrrophilic trap that interacts with the catalytic threonine at the N-terminus of mature 13-type subunits and a peptide moiety. A peptide epoxykerone YU-101 (Figure 2A) was the first highly potent and specific inhibitor of the chymotrypsin-like sites to be developed [7]. Carfilzomib is a derivative ofthis inh ibiror [3]. It is the most specific inhibitor of the chymotrypsin- like sites among FDA-approved agents or agents under- going clinical trials. NC-005 is even more specific than YU-101 and carfilzomib [8] whiJe IPSI-001 inhibits both 131i and 135i [9]. Further efforts ro improve selectivity have challenged the common dogma that active-site specificity is determined by the peptide portion of the inhibiror but not by the active-site elecrrophile. It was found that replacing epoxyketone in NC-005 (Figure 2A) with a vinyl sulfone moiety increases specificity for the chymotrypsin-like sites [10•]. Finally, utilization of

fluorinated amino acids had led ro another compound with improved specificity for the chymotrypsin-like activity, LU-005 (Figure 2A) [11].

Systematic modification of the peptide epoxykerone scaf- fold led to the development of the 135-specific inhibitor PR-825 (Figure 2F), and two 135i inhibitors, PR-924 and PR-957 (later renamed ONX-0914, Figure 2E) [12.,13 •• ].

Replacement of phenyl side chains in the P1 position in PR-924 and PR-957 with a cyclohexyl generated the highly selective 135i inhibitors LU-015i and LU-005i (Figure 2E) [14]. Oxathiazolones (Figure 2E) were first discovered as inhibitOrs of mycobacterial proteasomes but a recent study revealed that they show considerable selectivity roward 135i over 135 sites [15•]. The residues involved in the stabilization of the oxathiazolone covalent adduces with the mycobacterial proteasomes and the 135i site of human immunoproteasomes are similar. Oxathia- zolones have a short half-life in aqueous solutions [15•], which may limit their in vivo activity. Lascly, a number of non-peptide inhibitors of 135i were identified recencly by virtual screening [16].

YU-102 was the first inhibitOr of caspase-like sites to be developed [17], followed by the more specific NC-001 and compound 1 [18] (Figure 2C). All three epoxyketones inhibit both the 131 and 131i sites. Replacement of the isopropyl side chain in the P1 position with a cyclohexyl moiety, and fluorine substitutions in the 3-postion of the P3-Pro residue converted NC-001 into the 131i-specific inhibitor LU-001i (Figure 2D). LU-001i is more 131i- specific than another peptide epoxykerone UK-101 (Figure 2D), which also inhibitS 135i sites [14,19]. Dipep- tide boronate ML604440 is a more potent and specific inhjbitor of 131i sites [20]. All inhibitors are cell-per- meable.

A bigger challenge has been the development of cell- permeable inhibitors of the trypsin-like sites. The first specific inhibitors of these sites were cell impermeable [21,22]. Another structural class, peptide vinyl esters [23], irutially reported as ceiJ-permeable inhibirors of the tryp- sin-like sires, did not have any inhibitory activirywhen re- synthesized by another group [10•1. FinaJly two cell- permeable peptide epoxyketones, NC-002 and NC-022 (Figure 2B), were discovered a few years ago [24]. They were folJowed by the more potent vinyl sulfone LU-102 (Figure 2B), which also has a better cell permeability [25 •• ]. Just as with inhibirors of the chymotrypsin-like sites, these studies revealed that replacing the epoxyke- tone elecrrophiJe with vinyl sulfone dramatically increases the specificity of compounds for its primary target.

Most of these specific inhibitOrs were irutiaiJy used to define the role of different active sites as drug targets in cancer. For example, it was found that increasing the

specificityofcompoundsforthechymotrypsin-likesites dramatically decreases the cytotoxicity for HeLa cells [10]. Inhibitors of trypsin-like and caspase-like sites, whilenotcytotoxictocellswhenusedasasingleagent, sensitized multiple myeloma cells to inhibitors of the chymotrypsin-like sites, e.g. NC-005 and LU-005 [8].

Furthermore, inhibitors of the trypsin-like sites selec- tivelysensitizedmyelomacellstobortezomibandcarfil- zomib[24].Inotherwords,cytotoxicitywasobservedonly whentwotypesofactivesiteswereinhibited.Thelackof cytotoxiceffectofsite-specificinhibitorsoffersopportu- nitiesforusingthemfor immunomodulation.

Selectiveinhibitionoftheimmunoproteasome allows invivo modulation ofantigen

processingand T cell generation

When cells are stimulated with interferon (IFN)-g and tumor necrosis factor (TNF)-a, they produce vast

amountsof the inducibleb-type subunitsb1i,b2i, and b5i.Insuchapro-inflammatoryenvironmentvirtuallyall newly synthesized 20S proteasomes incorporate these induciblesubunitsratherthan theirconstitutivehomol- ogues b1, b2, and b5 to form immunoproteasomes. In miceinfected withviruses,bacteriaor fungiithasbeen shownthatintheliverandotherorgans(butnotthebrain [26]) constitutive proteasomes are largely replaced by immunoproteasomes[27,28].Theproteasomeiscentrally involved in the generation of peptide ligands of major histocompatibility complex (MHC) class I molecules which are recognized by theantigen receptors of cyto- toxic T lymphocytes (CTL) (Figure 3). Under stimu- lationwithIFN-gorTNF-athecellsurfaceexpressionof classImoleculesisup-regulated approximatelytenfold.

Theimmunoproteasomeisrequiredtoproducethissur- plusofclassIpeptideligandsneededbecausedeletionof b5i alone or all three immunosubunits (b1i, b2i, b5i)

Figure2

(a) β5β5i (e) β5i

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Current Opinion in Chemical Biology

Structuresofproteasomeinhibitorsmentionedinthisreviewgroupedaccordingtotheirproteasomesubunitspecificities.

Figure 3

some PA28

antigen.

presenting cell

Current Opinion in Chemical Biology

Antigen processing along the MHC class I pathway. Proteins synthesized in the cell are polyubiquitylated and degraded by the proteasome. Peptides produced are either of the ideal length for class I binding (8 9 amino acids) or are N terminally extended precursors which can be cleaved by amino peptidases in the cytoplasm. The transporter associated with antigen processing (TAP) transports the peptides into the endoplasmic reticulum (ER) where they can be further trimmed by ER amino peptidase (ERAP)1 /2. Peptides binding with high affinity to the MHC class I heavy chain/(32 microglobulin (b2m) complex induce a final folding and release from the ER lumenal chaperone calreticulin (in yellow) and the transmembrane chaperon calnexin (in green) to allow exit from the ER to the plasma membrane.

rogerher reduces class I surface expression in spleen, lymph nodes and thymus by approximately SO% [29].

Moreover, rhe trearmenr of cells or mice with rhe I3Si selective inhjbiror ONX-0914 (formerly called PR-957) leads ro a similar reduction in MHC class I surface expression indicating that I3Si is pivotal for the elevated generation of class I ligands [13••,30]. ONX-0914 exertS rhis effect in wild type but not in 135i_,_ mice indicating an on target effect of this first selective inhibiror of rhe immunoproteasome.

The mosr likely reason why immunoproteasomes pro- duce more or better suited class I ligands is rhat compared ro constitutive proreasomes they alter the cleavage pre- ferences such rhat class I ligands with higher binding affinities for the peptide binding groove of class I mol- ecules are generated. The caspase-like activity of 131, for example, produces weaker class I ligands than the chy- motrypsin-like activity of 131i because their C-rerminal anchor residues must possess hydrophobic or basic bur nor acidic side chains ro enable right binding imo the peptide cleft of class I molecules [31]. Since 135 and I3Si both exert a chymotrypsin-like activity, the requirement for this exchange was Jess obvious. However, the recently reported high resolution structures of mouse constitutive- and immunoproteasomes revealed that the 81 substrate

pocket of I3Si accommodates better bulky aliphatic and aromatic hydrophobic side chains [32••1 which are most frequently found in peptides eluted from class I mol- ecules. In spite of this valuable structural insight, the consequence of replacing 132 with 132i has remained elusive as they both harbor an equivalenr trypsin-like activity [33,34] and no obvious differences between the peptide binding pockets of 132 and 132i have been noted.

Accordingly, the generation of selective inhibirors for 132 and 132i will be a formjdable task.

The effect of immunoproteasome deficiency or inhibition on the bulk supply of MHC class I ligands on immune defence is limited. Mice lacking the inducible subunits are immunocompetenr except for an enhanced suscepti- biljry ro experimenraJ infections with Toxoplasma g01rdii [35] and Listeria mo11ocytoge11es [36]. However, on the level of rhe unique epitope the presence of immunoprotea- somes can be crucial either because the activity of 131i, 132i, or I3Si is required for generating the epitope [37] or because epitope destruction through the constitutive subunits 131, 132, or 135 needs ro be prevented by replace- ment with rhe respective immuno-suburuts [20]. The importanr role ofimmunoproteasome subunits in defirung the repertoire of peptides presenred on MHC class I molecules has been underlined by the mass spectrometric analysis of peprides eluted from rwo class I molecules of splenocytes of wild type and 131i!l32i/135i triply gene deleted mjce [29]. The advenr of suburut specific inhibi- tOrs has tl1erefore enabled the pharmacological modu- lation of peptide anrigen generation and presentation as well as the developmenr and expansion of cognate T cells itt vivo [13••,30]. Whether this potential of subunit se- lective proreasome inhibitors can be exploited for pharmacological intervenrion with infectious or auto- immune diseases remains to be determined.

lmmunoproteasome subunits as novel targets for the suppression of autoimmunity

Accumulating evidence indicates that rhe immunoprotea- some plays a cruciaJ role for the pathogenesis of several autoimmune diseases and that this role is unrelated to the MHC class I anrigen processing pathway. It was found that both, CDS+ CTLs and CD4+ T helper cells, need the immunoproteasome ro survive in a pro-inflammatory en- vironmenr [38]. Consequenrly, the inhibition of I3Si, 132i, or 131i may be a means to ameliorate pro-inflammatOry autoimmune diseases which rely on these cells. In fact, the treatmenr of mice with the I3Si selective inhibitor ONX-0914 prevenred rhe developmenr or progression of experimenral artluitis [13••1, diabetes [13 •• ], inflamma- tory bowel disease [39], systemic lupus erythematOsus [40], and experimental autoimmune encephalomyelitis (EAE) [41••1 in preclinical mouse models. Unexpectedly, I3Si-deficienr mice did not display the same protection profile as ONX-0914 treated wild type mjce in each ofthese models. Both, I3Si inhibition and deficiency protected mice

from dextrane sulfate sodium (DSS) induced colitis indicating that b5i plays an essential role in disease developmentwhichcannotbesustainedbyb5activity [39,42].InthemultiplesclerosismodelEAE,incontrast, protection from disease progression and relapse was observed in ONX-0914 treated wild type but not in b5i ^/ mice [41]. However, the treatment of b5i ^/ butnot wildtype micewith theb5-selectiveinhibitor PR-825suppressedEAE.Sinceb5completelyoccupies theb5/b5ipositionintheproteasomeofb5i ^/ mice,this result strongly suggests that inhibition with PR-825 suppressedthechymotrypsin-likeactivityinb5i ^/ mice to a degree which blocked the pathogenic cells. This interpretation is consistent with high immunoprotea- some expression in bone marrow-derived cells which causethedisease. Thelessonlearntfrom theseexper- imentsisthatb5iselectiveinhibitorsmaynotonlybeof therapeuticpotentialfordiseaseswhichrelyon anon- redundantfunction ofb5i butforall pro-inflammatory diseaseswhichrelyonleukocytesexpressinghighlevels ofimmunoproteasomes.

Atfirstsightonemayarguethatmoregeneralinhibitorsof the proteasomal chymotrypsin-like activity could be applied.However,immunoproteasomes arelargelycon- finedtositesofinflammationandleukocytesandthere- fore b5i selective therapy will block them and notthe majority of tissues which express mainly constitutive proteasomes. Consistently, it has been shown that ONX-0914is effectivetenfoldbelow itsmaximally tol- erateddosewhileinhibitorsofb5iandb5likebortezomib orcarfilzomibhavetobeappliedatthemaximallytoler- ateddose.Theresultingadverseeffectsareacceptablefor thetreatment ofneoplasticdiseaseslike multiplemyel- omaormantlecelllymphomabutareprobablytoosevere forthelongtermtreatmentof chronicautoimmunedis- eases.

Conclusion

The selective inhibition of proteasome subunits is an interestingapproachespeciallyifspecificsubunitsofthe immunoproteasomebutperhapsalsoofthethymoprotea- some[37,43]aretargeted.Thehighresolutionstructures of the 20S constitutive- and immunoproteasome have suggestedthatimmunoproteasomesshouldbedruggable andthefirstinhibitorof b5ihasproventheprinciplein preclinical models of autoimmunity. Immunoprotea- somesareexpressedinlymphocytes,antigenpresenting cellsandinnateimmunecellsanditwillbeimportantto elucidatewhichcellsareprimarilytargetedinwhichtype of disease. A similarlybig challengewill beto findout howtheimmunoproteasomeismechanisticallyinvolved on a molecular level in the pathogenesis especially in those disease models which rely on a non-redundant function of the immunoproteasome. Hopefully site specific inhibitors of the immunoproteasome will help toidentifyselectivesubstratesorcleavageactivitiesofthe

immunoproteasome. Based on the reported preclinical results research teams in academia and pharmaceutical industryhaveintensifiedthesearchforselectiveprotea- someinhibitorswhichwillhopefullysoonbesubjectedto clinicaltesting.

Conflictofinterest

Theauthors havenofinancial conflictof interest.

Acknowledgements

WorkdescribedinthisarticlewasfundedbytheGermanResearch FoundationgrantsGR1517/12-1andGR1517/14-1,theSwissNational ScienceFoundationgrant31003A 138451,andtheKonstanzGraduate SchoolChemicalBiology(toMG),aswellasbytheNorrisCottonCancer CenterNCIcoregrant(P30CA023108)andbyNCIgrant1R01-CA124634 (toAFK).

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