Miklós Békés

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.

SENPs are proteases that participate in the regulation of SUMOylation by generating mature small ubiquitin-related modifiers (SUMO) for protein conjugation (endopeptidase activity) and removing conjugated SUMO from targets (isopeptidase activity). Using purified recombinant catalytic domains of 6 of the 7 human SENPs, we demonstrate the specificity of their respective activities on SUMO-1, -2, and -3. The primary mode of recognition of substrates is via the SUMO domain, and the C-terminal tails direct endopeptidase specificity. Broadly speaking, SENP1 is the most efficient endopeptidase, whereas SENP2 and -5–7 have substantially higher isopeptidase than endopeptidase activities. We developed fluorogenic tetrapeptide substrates that are cleaved by SENPs, enabling us to characterize the environmental profiles of each enzyme. Using these synthetic substrates we reveal that the SUMO domain enhances catalysis of SENP1, -2, -5, -6, and -7, demonstrating substrate-induced activation of SENPs by SUMOs. SENPs are proteases that participate in the regulation of SUMOylation by generating mature small ubiquitin-related modifiers (SUMO) for protein conjugation (endopeptidase activity) and removing conjugated SUMO from targets (isopeptidase activity). Using purified recombinant catalytic domains of 6 of the 7 human SENPs, we demonstrate the specificity of their respective activities on SUMO-1, -2, and -3. The primary mode of recognition of substrates is via the SUMO domain, and the C-terminal tails direct endopeptidase specificity. Broadly speaking, SENP1 is the most efficient endopeptidase, whereas SENP2 and -5–7 have substantially higher isopeptidase than endopeptidase activities. We developed fluorogenic tetrapeptide substrates that are cleaved by SENPs, enabling us to characterize the environmental profiles of each enzyme. Using these synthetic substrates we reveal that the SUMO domain enhances catalysis of SENP1, -2, -5, -6, and -7, demonstrating substrate-induced activation of SENPs by SUMOs. Small ubiquitin-related modifier (SUMO) 3The abbreviations used are: SUMOsmall ubiquitin-like modifierAFC7-amino-4-trifluoromethyl coumarinDUBde-ubiquitinating enzymeSENPSentrin-specific proteaseDTTdithiothreitolNi-NTAnickel-nitrilotriacetic acidPIPES1,4-piperazinediethanesulfonic acidMES4-morpholineethanesulfonic acidCHES2-(cyclohexylamino)ethane-sulfonic acidCAPS3-(cyclohexylamino)propanesulfonic acidBistris propane1,3-bis[tris(hydroxymethyl)methylamino]propane. belongs to a family of ubiquitin-like proteins that, similar to ubiquitin, are conjugated to their substrates by a dedicated ligation system. Conjugation of SUMO in most cases results in altered subcellular localization of the modified protein, with consequent effects on its activity. The list of proteins subjected to SUMOylation is rapidly growing, and includes proteins localized in most subcellular compartments that are involved in the regulation of cell cycle, transcription, cell survival and death, DNA damage response, heat shock, and stress response, as well as endoplasmic reticulum and plasma membrane-associated proteins, receptors, and viral proteins (reviewed in Refs. 1Alarcon-Vargas D. Ronai Z. Cancer Biol. Ther. 2002; 1: 237-242Crossref PubMed Scopus (51) Google Scholar, 2Wilson V.G. Rosas-Acosta G. Sci. STKE 2005. 2005; : PE32Google Scholar, 3Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar). small ubiquitin-like modifier 7-amino-4-trifluoromethyl coumarin de-ubiquitinating enzyme Sentrin-specific protease dithiothreitol nickel-nitrilotriacetic acid 1,4-piperazinediethanesulfonic acid 4-morpholineethanesulfonic acid 2-(cyclohexylamino)ethane-sulfonic acid 3-(cyclohexylamino)propanesulfonic acid 1,3-bis[tris(hydroxymethyl)methylamino]propane. Modification of proteins by SUMO is a dynamic and reversible process. The SUMO cycle begins when SUMO precursors are processed to remove short C-terminal extensions, thereby uncapping the C-terminal Gly-Gly motif that is essential for conjugation. SUMO ligases conjugate the protein, via its C-terminal carboxylate, to the side-chain lysine of target proteins to generate an isopeptide linkage. Eventually, SUMO is removed intact from its substrate SUMOylated proteins, and so the SUMOylation/deSUMOylation cycle regulates SUMO function. A group of proteases known as SENPs are involved in both the maturation of SUMO precursors (endopeptidase cleavage) and deconjugation of the targets (isopeptidase cleavage) (4Melchior F. Schergaut M. Pichler A. Trends Biochem. Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 5Mukhopadhyay D. Dasso M. Trends Biochem. Sci. 2007; 32: 286-295Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Not all SENPs are SUMO-specific, indeed SENP8 is not a SUMO protease but functions on another small ubiquitin-related protein known as Nedd8 (6Gan-Erdene T. Nagamalleswari K. Yin L. Wu K. Pan Z.Q. Wilkinson K.D. J. Biol. Chem. 2003; 278: 28892-28900Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 7Wu K. Yamoah K. Dolios G. Gan-Erdene T. Tan P. Chen A. Lee C.G. Wei N. Wilkinson K.D. Wang R. Pan Z.Q. J. Biol. Chem. 2003; 278: 28882-28891Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The defining characteristic of SENPs is their predicted conserved molecular scaffold, defined as members of peptidase clan CE (8Rawlings N.D. Morton F.R. Barrett A.J. Nucleic Acids Res. 2006; 34: D270-D272Crossref PubMed Scopus (472) Google Scholar), conserved catalytic mechanism, and their reported activity on SUMO or Nedd8 conjugated proteins (or the respective precursors). Humans contain seven SENPs (SENPs -1, -2, -3, -5, -6, -7, and -8), and several of these have been characterized as SUMO (or Nedd8) endopeptidases or isopeptidases. However, there exists almost no information on SENP6 and -7, and there is considerable diversity in the literature regarding the specificity of individual SENPs for distinct SUMOs. In the effort to define the relative selectivities within the family, we have expressed and purified the catalytic domains corresponding to human SENP1, -2, and -5, -6, -7, and -8 and systematically analyzed their endopeptidase and isopeptidase activity on SUMO -1, -2, and -3 and Nedd8. This study also presents for the first time a fluorogenic assay based on simple peptides developed for SENPs that allow us to determine the influence of the SUMO targets on SENP activity and activation. Plasmid Constructs—Catalytic domains of SENP2, SENP7, and SENP8 were amplified from a human fetal brain cDNA library, and the catalytic domains of SENP1, SENP3, SENP5, and SENP6 were amplified from a human keratinocyte library. The PCR products were cloned into the bacterial expression vector pET28a (Novagen) engineered to contain an N-terminal His tag. The plasmids expressing SUMO precursor proteins His-tagged at the C terminus were generated using NcoI-XhoI restriction sites in pET28a. The NcoI restriction site incorporated a change from Ser at position 2 to Ala in SUMO-1 and SUMO-3 proteins, but SUMO-2 already contains Ala at this position. Because of some inconsistency in the literature regarding SUMO nomenclature (9Xu Z. Au S.W. Biochem. J. 2005; 386: 325-330Crossref PubMed Scopus (71) Google Scholar, 10Reverter D. Lima C.D. Structure. 2004; 12: 1519-1531Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Shen L. Dong C. Liu H. Naismith J.H. Hay R.T. Biochem. J. 2006; 397: 279-288Crossref PubMed Scopus (112) Google Scholar, 12Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar), in this study we define human pro-SUMO-2 as ending with the C-terminal extension VY and human pro-SUMO-3 with VPESSLAGHSF according to the HUGO nomenclature recommendations. The coding sequence for pro-Nedd8 was amplified from a human fetal brain library and inserted into the pET28a plasmid yielding the construct His6-Nedd8-His6. C-terminally truncated SUMO constructs were generated from full-length constructs and inserted into the pET28a vector in-frame with an N-terminal His tag. The cDNA coding for human RanGAP1ΔN (amino acids 418–588) was amplified by PCR from human fetal brain library and cloned into the mammalian expression vector pcDNA3 in-frame with an N-terminal FLAG sequence. A cDNA coding for Ubc-9 SUMO-conjugating enzyme was obtained by PCR from a human fetal brain library and inserted into pGEX-2T vector (GE Healthcare). All constructs were verified by DNA sequencing. Protein Expression in Escherichia coli—Recombinant SENP enzymes, SUMO proteins, and Nedd8 were produced in E. coli codon plus (Novagen). Production of SUMO proteins and Nedd8 was induced with 0.4 mm isopropyl β-d-galactopyranoside at 37 °C for 3 h. Expression of SENPs was induced with 0.2 mm isopropyl β-d-galactopyranoside at 30 °C for 3 h (for the expression of catalytic domains of SENP1, SENP2, SENP5, and SENP8) or at 25 °C for 5 h when expressing SENP6 and SENP7 catalytic domains. His-tagged proteins were purified using Ni-NTA-agarose and eluted with a 20–200 mm gradient of imidazole in 50 mm HEPES, pH 7.4, 100 mm NaCl. GST-Ubc9 was expressed in E. coli BL21 (DE3) under standard conditions provided by the manufacturer (Novagen), purified by affinity chromatography using glutathione-Sepharose, and eluted with 10 mm reduced glutathione in 50 mm Tris, pH 8.0. Protein purity was examined by SDS-PAGE, and concentrations of the purified proteins were determined from the absorbance at 280 nm based on the molar absorption coefficients determined from the Edelhoch relationship (13Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3064) Google Scholar). Tissue Culture, Transfection, and Protein Purification—HEK293A cells were grown in Dulbecco's modified Eagle's medium, containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine. The cDNA constructs FLAG-RanGAP1ΔN was used to transfect HEK293A cells using GeneJuice (Novagen) as recommended by the manufacturer. 36 h after transfection, cells were in and on in a containing 50 mm HEPES, mm with the protease and G. H. A Scholar). FLAG-RanGAP1ΔN was to affinity at and were in and eluted with of μg/ml In generate mature SUMO proteins ending with a Gly-Gly the corresponding precursor proteins were in 50 mm Tris, pH mm 2 mm with SENP1 enzyme for 2 h at 37 cleaved C-terminal and enzyme all contain His and were removed from the using The mature SUMO proteins used for in SUMOylation In SUMOylation were in a of 100 of containing mm HEPES, pH 7.4, 5 mm of GST-Ubc9 at 50 μg/ml of 10 of the respective mature SUMO at 50 10 of mm and 3 of E. Naismith J.H. Hay R.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 2 h at 37 °C the SUMOylation was of Ubc-9 enzyme by using The was used as a of SUMOylated SENP and used an gradient for protein A. J. Scopus Google Scholar). in the of and pro-Nedd8 we that PubMed Scopus Google Scholar). the of SENPs to SUMO-1, or Nedd8 substrate was with purified recombinant at the concentrations at 37 °C for h in containing 50 mm Tris, pH mm 5 mm and the was with were subjected to by The of precursor protein was from the of and this was enzyme to the of enzyme to of precursor isopeptidase activity of SENPs, 5 of SUMOylated was with concentrations of enzyme in for h at 37 products were on and by The of SUMO from RanGAP1ΔN was determined by primary by and was from the by a using or by on and The are the of SENP on tetrapeptide substrates corresponding to the site in the SUMO and Nedd8 protein substrates were as on the results from a substrate library, J. K. Z. and G. for we used for SENP1, -2, and and for SENP -6, -7, and The of these substrates J. K. Z. and G. for of recombinant SENPs was in 50 mm Tris, pH mm 5 mm and in mm Tris, pH 5 mm enzyme assay were at 37 °C in the 100 substrate and enzyme concentrations and 6 of was using an determine the catalytic of the were as a of substrate substrate concentrations from to were the of a with The were using concentrations were as of pH and on SENP at 50 mm were for the pH for the pH pH for the pH pH for the pH pH for the pH pH for the pH pH and at pH The also mm and 5 mm the of or on SENP 50 mm Tris, pH 5 mm and the of Expression of engineered for the catalytic domains of SENP1, SENP2, and were based on the expression of these proteins in E. coli D. Lima C.D. Structure. 2004; 12: 1519-1531Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Shen L. Dong C. Liu H. Naismith J.H. Hay R.T. Biochem. J. 2006; 397: 279-288Crossref PubMed Scopus (112) Google Scholar, A. J. Lee A. H. G. Biol. 2006; PubMed Scopus Google Scholar), with The of SENP3, and SENP7 catalytic domains was based on a using 34: PubMed Scopus Google Scholar), to the SENP2 catalytic domain, of the characterized members of the family from a D. Lima C.D. Structure. 2004; 12: 1519-1531Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, D. Lima C.D. Biol. 2006; PubMed Scopus Google Scholar). SENP8 not have an N-terminal and we the full-length The domains were expressed in E. coli as N-terminal His-tagged proteins and purified using proteins in with corresponding to the predicted molecular SENP1, -2, and -8 of protein of E. coli and purified was substantially SENP -5, -6, and were expressed at of E. coli and the in some E. coli proteins the of SENP6 and The construct and construct proteins, but of the produced a recombinant However, similar for in of an protein, and we were to characterize All recombinant SENPs used in this study as with the in that the constructs are not have of activity. the precursors of by removing C-terminal extensions, as endopeptidase activity. In SUMO to target proteins, as isopeptidase activity. SENP1, -2, -3, -5, and have each been to endopeptidase activity on at of the human SUMO precursors A. J. Lee A. H. G. Biol. 2006; PubMed Scopus Google Scholar, L. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, H. H. Biol. 2002; PubMed Scopus Google Scholar, T. H. H. J. Biochem. 2000; PubMed Scopus Google Scholar, D. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and on this we that is a substrate for SENP7, this not been We determined the of each SENP in of its endopeptidase and isopeptidase activities by the of enzyme at a substrate We used as endopeptidase substrates and the in SUMOylated RanGAP1ΔN as an isopeptidase the of SENPs on the distinct we were to demonstrate in specificity within the family 2 and SENP1 as by the most efficient endopeptidase of the family, and SENP2 and similar but endopeptidase activity. In SENP2 and were almost as efficient as SENP6 and were almost endopeptidase activity. also isopeptidase this was higher than their endopeptidase activity and pro-SUMO-3 was an endopeptidase substrate but was as as SUMO-2 and was than SUMO-1 for all SENPs SENP8 no activity on but of pro-Nedd8 The of mature SUMO-2 and -3 are but with to the that the in the activity of SENP2, -5, -6, and for pro-SUMO-2 pro-SUMO-3 by the sequence in the C-terminal been for SENP1 and SENP2 that the of on the corresponding to these (9Xu Z. Au S.W. Biochem. J. 2005; 386: 325-330Crossref PubMed Scopus (71) Google Scholar, 10Reverter D. Lima C.D. Structure. 2004; 12: 1519-1531Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). We the of the tails by pro-SUMO-2 and pro-SUMO-3 to SUMO-3 with the SUMO-2 and this In each SENP the was a substrate than pro-SUMO-3 for almost the activity on the substrate pro-SUMO-2 This that, SUMO-2 and SUMO-3 a of endopeptidase using with to the We that the C-terminal tails a in defining endopeptidase specificity of SENPs in the of pro-SUMO-2 and of SENPs on this all and of SENP activity been defined using full-length SUMO However, there is no a to that short synthetic used to define protease not used with SUMO and SENPs, the catalytic is in a similar to proteases from that are on short and the of SENP1, -2, and as well as the of the have defined site D. Lima C.D. Structure. 2004; 12: 1519-1531Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Shen L. Dong C. Liu H. Naismith J.H. Hay R.T. Biochem. J. 2006; 397: 279-288Crossref PubMed Scopus (112) Google Scholar, Z. Au S.W. Biochem. J. 2006; PubMed Scopus Google Scholar, E. Lima C.D. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. Wu K. Pan Z.Q. Wilkinson K.D. Lima C.D. J. Biol. 2005; PubMed Scopus Google Scholar). we substrates to define the relative of the site and the substrate and enzyme. the of fluorogenic substrates us to define catalytic of the We the substrates based on the of and with an fluorogenic the C-terminal tails In this we were to assay enzyme activity of SENP1 and on and SENP8 on Using the no activity was for SENP5, -6, and on of the determine the conditions in SENPs the of a of was a of pH and concentrations The activity of SENP1, -2, and -8 from pH a at pH after there was a The of the was pH is with of the catalytic in the of proteases A. and H. Scholar). concentrations of the a on activity of SENP1 and SENP2 than for by simple as by the of of that a the activity of SENPs several of is that the site of the enzyme is not in an for the the as for the protease G. M. L. Biochemistry. PubMed Scopus Google Scholar), of on the the site and catalysis in a similar to the of the SUMO of SENP1 L. Dong C. Liu H. Naismith J.H. Hay R.T. Biochem. J. 2006; 397: 279-288Crossref PubMed Scopus (112) Google and SENP2 D. Lima C.D. Structure. 2004; 12: 1519-1531Abstract Full Text Full Text PDF PubMed Scopus (158) Google with or SUMO substrates that the small substrate The of synthetic substrates us to these on enzyme activity. SUMO not enzyme is of the C terminus of SUMO and the synthetic substrate that in the enzyme this the of we acids from the C terminus of mature SUMO-1, and -3 the respective to this to the tetrapeptide substrate at the site A and of all SENPs was by at of the truncated and of SENPs was at molar of truncated and the that enzyme activity was We used the for SENP1 and and for SENP5, -6, and SENP1 and not and enzyme activity was induced almost by truncated SENP2, -6, and -7, on the and the of on enzyme activity were at mm the the of truncated SUMO not that in are in SUMO and activation. In to the SENPs, the SENP8 was by and truncated Nedd8 no In this study we the of members of the human SENP family in of their endopeptidase and isopeptidase specificity. We for the first time their activity on small synthetic us to substrate-induced activation for some members of the family, as well as a of their for activity. are to we on of in from expression in E. was not in study is in some we no in the protein, is a we expressed the C-terminal catalytic domains and not full-length proteins, with the of the enzyme not contain an N-terminal The N-terminal of SENPs influence the of the in and influence their as However, there is no to influence the of the contain no of known protein in us to the of the conserved catalytic domains from the In of endopeptidase activity on SUMO are with for SENP1 (9Xu Z. Au S.W. Biochem. J. 2005; 386: 325-330Crossref PubMed Scopus (71) Google and SENP2 D. Lima C.D. Structure. 2004; 12: 1519-1531Abstract Full Text Full Text PDF PubMed Scopus (158) Google that SENP1 pro-SUMO-2 whereas SENP2 pro-SUMO-2 substrate almost to literature is regarding the relative of SENP5, with regarding the of the enzyme A. J. Lee A. H. G. Biol. 2006; PubMed Scopus Google Scholar, L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). This is of of the C-terminal demonstrate that is to SENP2 in its endopeptidase and isopeptidase activities. SENP6 and were substantially as and in cleaved pro-SUMO-2 at a This is with the that SENP6 not in as a enzyme D. F. N. Tan T. A. Wilkinson K.D. Dasso M. J. Biol. 2006; PubMed Scopus Google Scholar), and we that the is for The isopeptidase using as in SENP specificity. SUMO-3 a substrate when conjugated to than when is a This was using recombinant enzyme and and also in cell containing SENP activity. in to SENP1, all SENPs are than are This that SENP1 the most SUMO endopeptidase in is with its (9Xu Z. Au S.W. Biochem. J. 2005; 386: 325-330Crossref PubMed Scopus (71) Google Scholar). the most that the SENP6 and -7, from members of the family is the of the for This a to the that of the Gly-Gly of SUMO to the site of SENPs, and its by to Ala in SENP2 activity D. Lima C.D. Biol. 2006; PubMed Scopus Google Scholar). at this position endopeptidase activity but isopeptidase and the C-terminal tails of are of specificity as by the the tails of pro-SUMO-2 and -3, and isopeptidase endopeptidase activity. this was for SENP1 and of the tails of (9Xu Z. Au S.W. Biochem. J. 2005; 386: 325-330Crossref PubMed Scopus (71) Google Scholar, 10Reverter D. Lima C.D. Structure. 2004; 12: 1519-1531Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), and we this is a of all the SENPs the C-terminal is the primary that SENPs to SUMO-2 and -3. The of the sequence of the C-terminal the results of on A. J. Lee A. H. G. Biol. 2006; PubMed Scopus Google Scholar, L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). used constructs to the tails of to endopeptidase and we obtained are no regarding the of the pro-Nedd8 and SENP8 specificity. In this study for the first time we the of synthetic substrates to assay activity of The sequence used as a for the fluorogenic the recognition motif in all and the recognition motif in Nedd8. of the synthetic substrates were of than the corresponding proteins, the of the SUMO domain in substrate A similar been when the de-ubiquitinating isopeptidase and were on tetrapeptide fluorogenic substrates full-length conjugated to a C-terminal fluorogenic group Biochemistry. PubMed Scopus Google Scholar, M. A. R. D. M. Structure. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the we were to the small substrates to demonstrate the influence of pH and on SENP activity. The a pH of mammalian proteases and an of activity in concentrations of the This is of protein or domain G. M. L. Biochemistry. PubMed Scopus Google Scholar), a small of of the site in SENP2 when are with D. Lima C.D. Structure. 2004; 12: 1519-1531Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). us to that of SENPs with the enzyme to to The of enzyme in the of truncated the of but was than the of substrate that the substrate from SUMO plus the tetrapeptide sequence from the synthetic not allow for with the enzyme. In substrate in SUMO-1 and in SUMO-2 and are the not to with the enzyme in the the of a from truncated SUMO with the group at the terminus of the synthetic the of the C-terminal is for substrate and catalysis of the synthetic The of truncated to substantially activity is for substrate-induced a not reported for members of this family but in some members of the of M. A. R. D. M. Structure. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, M. P. M. T. Wu 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, F. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, H. R. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C. D. Sci. A. 2006; PubMed Scopus Google Scholar, J. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The substrate activation of and activation of SENPs is that, with to synthetic substrates that the site of the enzymes, full-length Biochemistry. PubMed Scopus Google Scholar), but full-length SUMO not In the the catalytic are when are with the enzyme is in the of A in the that the in SENP2 is in D. Lima C.D. Biol. 2006; PubMed Scopus Google Scholar), but the and of SENP1 and both to have catalytic in the activation of SENP1, -2, and -5, -6, and is truncated Nedd8 not and the is not from the the substrate-induced activation have another in SENPs, or the not the protein in SENP8 a of substrate-induced activation by This us of and SENP8 reveal a in the that the substrate to the the change within the -2, and SENPs tetrapeptide substrates with and the human contains or that proteins SENPs have a the SUMO domain enhances thereby for SUMO A specificity by all SENPs is at the C-terminal of the are the almost SUMO-2 and -3. results reveal no SUMO-2 and -3 when to the conjugation target but endopeptidase based on the distinct C-terminal and from this is that the sequence of the SUMO-2 is conserved in but there is no in sequence or of the SUMO-3 the of the C-terminal we that not all SUMOylation sites in target proteins by the the to of the regulation of specificity. We for with

PURPOSE: Estrogen receptor (ER) alpha signaling is a known driver of ER-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Combining endocrine therapy (ET) such as fulvestrant with CDK4/6, mTOR, or PI3K inhibitors has become a central strategy in the treatment of ER+ advanced breast cancer. However, suboptimal ER inhibition and resistance resulting from the ESR1 mutation dictates that new therapies are needed. EXPERIMENTAL DESIGN: A medicinal chemistry campaign identified vepdegestrant (ARV-471), a selective, orally bioavailable, and potent small molecule PROteolysis-TArgeting Chimera (PROTAC) degrader of ER. We used biochemical and intracellular target engagement assays to demonstrate the mechanism of action of vepdegestrant, and ESR1 wild-type (WT) and mutant ER+ preclinical breast cancer models to demonstrate ER degradation-mediated tumor growth inhibition (TGI). RESULTS: Vepdegestrant induced ≥90% degradation of wild-type and mutant ER, inhibited ER-dependent breast cancer cell line proliferation in vitro, and achieved substantial TGI (87%-123%) in MCF7 orthotopic xenograft models, better than those of the ET agent fulvestrant (31%-80% TGI). In the hormone independent (HI) mutant ER Y537S patient-derived xenograft (PDX) breast cancer model ST941/HI, vepdegestrant achieved tumor regression and was similarly efficacious in the ST941/HI/PBR palbociclib-resistant model (102% TGI). Vepdegestrant-induced robust tumor regressions in combination with each of the CDK4/6 inhibitors palbociclib, abemaciclib, and ribociclib; the mTOR inhibitor everolimus; and the PI3K inhibitors alpelisib and inavolisib. CONCLUSIONS: Vepdegestrant achieved greater ER degradation in vivo compared with fulvestrant, which correlated with improved TGI, suggesting vepdegestrant could be a more effective backbone ET for patients with ER+/HER2- breast cancer.

Deubiquitinating enzymes (DUBs) constitute a large family of cysteine proteases that have a broad impact on numerous biological and pathological processes, including the regulation of genomic stability. DUBs are often assembled onto multiprotein complexes to assist in their localization and substrate selection, yet it remains unclear how the enzymatic activity of DUBs is modulated by intracellular signals. Herein, we show that bursts of reactive oxygen species (ROS) reversibly inactivate DUBs through the oxidation of the catalytic cysteine residue. Importantly, USP1, a key regulator of genomic stability, is reversibly inactivated upon oxidative stress. This, in part, explains the rapid nature of PCNA monoubiquitination-dependent DNA damage tolerance in response to oxidative DNA damage in replicating cells. We propose that DUBs of the cysteine protease family act as ROS sensors in human cells and that ROS-mediated DUB inactivation is a critical mechanism for fine-tuning stress-activated signaling pathways.