ABSTRACT Covalent modulators and covalent degrader molecules have emerged as drug modalities with tremendous therapeutic potential. Toward realizing this potential, mass spectrometry-based

chemoproteomic screens have generated proteome-wide maps of potential druggable cysteine residues. However, beyond these direct cysteine-target maps, the full scope of direct and indirect

activities of these molecules on cellular processes and how such activities contribute to reported modes of action, such as degrader activity, remains to be fully understood. Using

chemoproteomics, we identified a cysteine-reactive small molecule degrader of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 14 (nsp14), which effects

we delineate the wide-ranging impacts of cysteine-reactive electrophilic compounds on cellular proteostasis processes. Access through your institution Buy or subscribe This is a preview of

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A COVALENT CHEMICAL PROBE FOR _CHIKUNGUNYA_ NSP2 CYSTEINE

PROTEASE WITH ANTIALPHAVIRAL ACTIVITY AND PROTEOME-WIDE SELECTIVITY Article Open access 01 March 2025 TARGETED DEGRADATION OF EXTRACELLULAR MITOCHONDRIAL ASPARTYL-TRNA SYNTHETASE MODULATES

IMMUNE RESPONSES Article Open access 22 July 2024 DEUBIQUITINASE-TARGETING CHIMERAS FOR TARGETED PROTEIN STABILIZATION Article 24 February 2022 DATA AVAILABILITY All supporting data for this

study can be found within the article and Supplementary Information. The MS data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the

Proteomics Identification Database (PRIDE)99 partner repository with the dataset identifiers PXD046278 (isoTOP–ABPP data in Figs. 1 and 5), PXD046393 (all other proteomic data corresponding

to Figs. 3–6) and PXD053865 (PDIA6-His AP–MS and Extended Data Fig. 4 proteomics). Publicly available databases used are the UniProtKB Consortium (https://www.uniprot.org/), the 2021 release

of the KEGG pathway database (https://www.genome.jp/kegg/pathway.html) and the 2021 release of the GO Resource (https://geneontology.org/) Molecular Function, Cellular Component and

Biological Process classes. Source data are provided as source data files for Figs. 1–6 and Extended Data Figs. 2, 3, 4, 7 and 9 and within the Supplementary Information for Supplementary

Figs. 1–30. Source data are provided with this paper. CODE AVAILABILITY All code used for this work is available at https://github.com/BackusLab. REFERENCES * Boatner, L. M., Palafox, M. F.,

Schweppe, D. K. & Backus, K. M. CysDB: a human cysteine database based on experimental quantitative chemoproteomics. _Cell Chem. Biol._ 30, 683–698 (2023). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Backus, K. M. et al. Proteome-wide covalent ligand discovery in native biological systems. _Nature_ 534, 570–574 (2016). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. _Nature_ 468, 790–795 (2010). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Cao, J. et al. Multiplexed CuAAC Suzuki–Miyaura labeling for tandem activity-based chemoproteomic profiling. _Anal. Chem._ 93, 2610–2618 (2021). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Vinogradova, E. V. et al. An activity-guided map of electrophile–cysteine interactions in primary human T cells. _Cell_ 182, 1009–1026 (2020). Article  CAS

  PubMed  PubMed Central  Google Scholar  * Yan, T. et al. SP3-FAIMS chemoproteomics for high-coverage profiling of the human cysteinome. _ChemBioChem_ 22, 1841–1851 (2021). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Kuljanin, M. et al. Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries. _Nat.

Biotechnol._ 39, 630–641 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Burton, N. R. et al. Solid-phase compatible silane-based cleavable linker enables custom isobaric

quantitative chemoproteomics. _J. Am. Chem. Soc._ 145, 21303–21318 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mader, M. M. et al. Which small molecule? Selecting

chemical probes for use in cancer research and target validation. _Cancer Discov._ 13, 2150–2165 (2023). Article  PubMed  Google Scholar  * Antolin, A. A. et al. The Chemical Probes Portal:

an expert review-based public resource to empower chemical probe assessment, selection and use. _Nucleic Acids Res._ 51, D1492–D1502 (2023). Article  PubMed  Google Scholar  * Hartung, I.

V., Rudolph, J., Mader, M. M., Mulder, M. P. C. & Workman, P. Expanding chemical probe space: quality criteria for covalent and degrader probes. _J. Med. Chem._ 66, 9297–9312 (2023).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Kavanagh, M. E. et al. Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine. _Nat. Chem. Biol._ 18,

1388–1398 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A. & Shokat, K. M. K-Ras(G12C) inhibitors allosterically

control GTP affinity and effector interactions. _Nature_ 503, 548–551 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Janes, M. R. et al. Targeting KRAS mutant cancers with a

covalent G12C-specific inhibitor. _Cell_ 172, 578–589.e17 (2018). Article  CAS  PubMed  Google Scholar  * Dickson, P. et al. Physical and functional analysis of the putative rpn13 inhibitor

RA190. _Cell Chem. Biol._ 27, 1371–1382 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Anchoori, R. K. et al. A bis-benzylidine piperidone targeting proteasome ubiquitin

receptor RPN13/ADRM1 as a therapy for cancer. _Cancer Cell_ 24, 791–805 (2013). Article  CAS  PubMed  Google Scholar  * Gamayun, I. et al. Eeyarestatin compounds selectively enhance

Sec61-mediated Ca2+ leakage from the endoplasmic reticulum. _Cell Chem. Biol._ 26, 571–583 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang, K. et al. Stress granule

assembly disrupts nucleocytoplasmic transport. _Cell_ 173, 958–971.e17 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Othumpangat, S., Kashon, M. & Joseph, P. Sodium

arsenite-induced inhibition of eukaryotic translation initiation factor 4E (eIF4E) results in cytotoxicity and cell death. _Mol. Cell. Biochem._ 279, 123–131 (2005). Article  CAS  PubMed 

Google Scholar  * Kopito, R. R. Aggresomes, inclusion bodies and protein aggregation. _Trends Cell Biol._ 10, 524–530 (2000). Article  CAS  PubMed  Google Scholar  * García-Mata, R., Bebök,

Z., Sorscher, E. J. & Sztul, E. S. Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera. _J. Cell Biol._ 146, 1239–1254 (1999). Article  PubMed  PubMed Central

  Google Scholar  * Forte, N. et al. Targeted Protein Degradation through E2 Recruitment. _ACS Chem. Biol._ 18, 897–904 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang,

X., Crowley, V. M., Wucherpfennig, T. G., Dix, M. M. & Cravatt, B. F. Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. _Nat. Chem. Biol._ 15, 737–746 (2019).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Tao, Y. et al. Targeted protein degradation by electrophilic PROTACs that stereoselectively and site-specifically engage DCAF1. _J.

Am. Chem. Soc._ 144, 18688–18699 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang, X. et al. DCAF11 supports targeted protein degradation by electrophilic proteolysis

targeting chimeras. _J. Am. Chem. Soc._ 143, 5141–5149 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Spradlin, J. N. et al. Harnessing the anti-cancer natural product

nimbolide for targeted protein degradation. _Nat. Chem. Biol._ 15, 747–755 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ward, C. C. et al. Covalent ligand screening

uncovers a RNF4 E3 ligase recruiter for targeted protein degradation applications. _ACS Chem. Biol._ 14, 2430–2440 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Henning, N.

J. et al. Discovery of a covalent FEM1B recruiter for targeted protein degradation applications. _J. Am. Chem. Soc._ 144, 701–708 (2022). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Isobe, Y. et al. Manumycin polyketides act as molecular glues between UBR7 and P53. _Nat. Chem. Biol._ 16, 1189–1198 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar 

* Toriki, E. S. et al. Rational chemical design of molecular glue degraders. _ACS Cent. Sci._ 9, 915–926 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sarott, R. C. et al.

Chemical specification of E3 ubiquitin ligase engagement by cysteine-reactive chemistry. _J. Am. Chem. Soc._ 145, 21937–21944 (2023). Article  CAS  PubMed  Google Scholar  * Li, Y.-D. et al.

Template-assisted covalent modification underlies activity of covalent molecular glues. _Nat. Chem. Biol._ https://doi.org/10.1038/s41589-024-01668-4 (2024). * Hassan, M. M. et al.

Exploration of the tunability of BRD4 degradation by DCAF16 _trans_-labelling covalent glues. _Eur. J. Med. Chem._ 279, 116904 (2024). Article  CAS  PubMed  PubMed Central  Google Scholar  *

King, E. A. et al. Chemoproteomics-enabled discovery of a covalent molecular glue degrader targeting NF-κB. _Cell Chem. Biol._ 30, 394–402 (2023). Article  CAS  PubMed  Google Scholar  *

Hong, S. H. et al. Exploiting the cullin E3 ligase adaptor protein SKP1 for targeted protein degradation. _ACS Chem. Biol._ 19, 442–450 (2024). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Littler, D. R., Gully, B. S., Colson, R. N. & Rossjohn, J. Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9. _iScience_ 23, 101258 (2020). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Ma, Y. et al. Structural basis and functional analysis of the SARS coronavirus nsp14–nsp10 complex. _Proc. Natl Acad. Sci. USA_ 112, 9436–9441

(2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Decroly, E. et al. Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2′-O-methyltransferase

nsp10/nsp16 complex. _PLoS Pathog._ 7, e1002059 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Imprachim, N., Yosaatmadja, Y. & Newman, J. A. Crystal structures and

fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development. _Nucleic Acids Res._ 51,

475–487 (2023). Article  CAS  PubMed  Google Scholar  * Liby, K. et al. The synthetic triterpenoids, CDDO and CDDO-imidazolide, are potent inducers of heme oxygenase-1 and Nrf2/ARE

signaling. _Cancer Res._ 65, 4789–4798 (2005). Article  CAS  PubMed  Google Scholar  * Yang, D. et al. Systematic targeting of protein complexes with molecular COUPLrs. Preprint a _bioRxiv_

https://doi.org/10.1101/2024.07.16.603666 (2024). * Mauvezin, C. & Neufeld, T. P. Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and

Ca-P60A/SERCA-dependent autophagosome-lysosome fusion. _Autophagy_ 11, 1437–1438 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ohtake, F., Tsuchiya, H., Saeki, Y. &

Tanaka, K. K63 ubiquitylation triggers proteasomal degradation by seeding branched ubiquitin chains. _Proc. Natl Acad. Sci. USA_ 115, E1401–E1408 (2018). Article  CAS  PubMed  PubMed Central

  Google Scholar  * Gordon, D. E. et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. _Nature_ 583, 459–468 (2020). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Grantham, J. The molecular chaperone CCT/TRiC: an essential component of proteostasis and a potential modulator of protein aggregation. _Front. Genet._ 11, 172 (2020). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Bashore, C. et al. Targeted degradation via direct 26S proteasome recruitment. _Nat. Chem. Biol._ 19, 55–63 (2023). Article  CAS  PubMed 

Google Scholar  * Tofaris, G. K., Layfield, R. & Spillantini, M. G. α-Synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome. _FEBS Lett._

509, 22–26 (2001). Article  CAS  PubMed  Google Scholar  * Zhou, M. et al. HUWE1 amplifies ubiquitin modifications to broadly stimulate clearance of proteins and aggregates. Preprint at

_bioRxiv_ https://doi.org/10.1101/2023.05.30.542866 (2023). * Sarkar, A. A. & Zohn, I. E. Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior

of the cranial mesenchyme. _J. Cell Biol._ 196, 789–800 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang, Q., Li, L. & Ye, Y. Inhibition of p97-dependent protein

degradation by eeyarestatin I. _J. Biol. Chem._ 283, 7445–7454 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Berkers, C. R. et al. Profiling proteasome activity in tissue

with fluorescent probes. _Mol. Pharm._ 4, 739–748 (2007). Article  CAS  PubMed  Google Scholar  * Leestemaker, Y. et al. Proteasome activation by small molecules. _Cell Chem. Biol._ 24,

725–736 (2017). Article  CAS  PubMed  Google Scholar  * Vetma, V. et al. Confounding factors in targeted degradation of short-lived proteins. _ACS Chem. Biol._ 19, 1484–1494 (2024). Article

  CAS  PubMed  Google Scholar  * Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. _Cell_ 149, 1060–1072 (2012). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Higgins, L. G. et al. Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of

electrophiles, peroxides and redox-cycling agents. _Toxicol. Appl. Pharmacol._ 237, 267–280 (2009). Article  CAS  PubMed  Google Scholar  * Hetz, C. The unfolded protein response:

controlling cell fate decisions under ER stress and beyond. _Nat. Rev. Mol. Cell Biol._ 13, 89–102 (2012). Article  CAS  PubMed  Google Scholar  * Edman, J. C., Ellis, L., Blacher, R. W.,

Roth, R. A. & Rutter, W. J. Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. _Nature_ 317, 267–270 (1985). Article  CAS  PubMed  Google

Scholar  * Plate, L. et al. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation. _eLife_ 5, e15550 (2016). Article  PubMed  PubMed

Central  Google Scholar  * Hoffstrom, B. G. et al. Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins. _Nat. Chem. Biol._ 6, 900–906 (2010). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Jean, S., Cox, S., Nassari, S. & Kiger, A. A. Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote

autophagosome-lysosome fusion. _EMBO Rep._ 16, 297–311 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Liu, Y. et al. A novel mechanism for NF-κB activation via IκB

aggregation: Implications for hepatic Mallory-Denk-body induced inflammation. _Mol. Cell. Proteomics_ 19, 1968–1986 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Yang, P.

et al. G3BP1 is a tunable switch that triggers phase separation to assemble stress granules. _Cell_ 181, 325–345 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Dinkova-Kostova, A. T. et al. Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress. _Proc. Natl Acad. Sci. USA_

102, 4584–4589 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  * Liu, Y. et al. Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential

therapeutic reagent for Huntington’s disease. _J. Neurochem._ 129, 539–547 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gromer, S., Arscott, L. D., Williams, C. H.,

Schirmer, R. H. & Becker, K. Human placenta thioredoxin reductase. Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds. _J. Biol. Chem._

273, 20096–20101 (1998). Article  CAS  PubMed  Google Scholar  * Zhang, J. et al. Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway.

_Cell_ 186, 2361–2379 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mao, X. et al. Novel multi-targeted ErbB family inhibitor afatinib blocks EGF-induced signaling and

induces apoptosis in neuroblastoma. _Oncotarget_ 8, 1555–1568 (2017). Article  PubMed  Google Scholar  * Huang, F. et al. Repurposing of ibrutinib and quizartinib as potent inhibitors of

necroptosis. _Commun. Biol._ 6, 972 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kapoor, I. et al. Resistance to BTK inhibition by ibrutinib can be overcome by preventing

FOXO3a nuclear export and PI3K/AKT activation in B-cell lymphoid malignancies. _Cell Death Dis._ 10, 924 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Lanning, B. R. et al.

A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors. _Nat. Chem. Biol._ 10, 760–767 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Trendel,

J. et al. The human RNA-binding proteome and its dynamics during translational arrest. _Cell_ 176, 391–403 (2019). Article  CAS  PubMed  Google Scholar  * Wang, X. et al. Selective depletion

of mutant p53 by cancer chemopreventive isothiocyanates and their structure-activity relationships. _J. Med. Chem._ 54, 809–816 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar

  * Yasuda, S. et al. Stress- and ubiquitylation-dependent phase separation of the proteasome. _Nature_ 578, 296–300 (2020). Article  CAS  PubMed  Google Scholar  * Gwon, Y. et al.

Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner. _Science_ 372, eabf6548 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Johnston, J.

A., Ward, C. L. & Kopito, R. R. Aggresomes: a cellular response to misfolded proteins. _J. Cell Biol._ 143, 1883–1898 (1998). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Zhang, C. et al. Autophagic sequestration of SQSTM1 disrupts the aggresome formation of ubiquitinated proteins during proteasome inhibition. _Cell Death Dis._ 13, 615 (2022). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Yu, C. et al. Afatinib combined with anti-PD1 enhances immunotherapy of hepatocellular carcinoma via ERBB2/STAT3/PD-L1 signaling. _Front. Oncol._

13, 1198118 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, D. et al. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models.

_Oncogene_ 27, 4702–4711 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bar-Peled, L. et al. Chemical proteomics identifies druggable vulnerabilities in a genetically

defined cancer. _Cell_ 171, 696–709 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Abegg, D. et al. Proteome-wide profiling of targets of cysteine reactive small molecules

by using ethynyl benziodoxolone reagents. _Angew. Chem._ 127, 11002–11007 (2015). Article  Google Scholar  * Grossman, E. A. et al. Covalent ligand discovery against druggable hotspots

targeted by anti-cancer natural products. _Cell Chem. Biol._ 24, 1368–1376 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang, C., Weerapana, E., Blewett, M. M. &

Cravatt, B. F. A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles. _Nat. Methods_ 11, 79–85 (2014). Article  PubMed  Google Scholar  * Henning, N. J. et

al. Deubiquitinase-targeting chimeras for targeted protein stabilization. _Nat. Chem. Biol._ 18, 412–421 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Lazear, M. R. et al.

Proteomic discovery of chemical probes that perturb protein complexes in human cells. _Mol. Cell_ 83, 1725–1742 (2023). Article  CAS  PubMed  Google Scholar  * Kathman, S. G. et al.

Remodeling oncogenic transcriptomes by small molecules targeting NONO. _Nat. Chem. Biol._ 19, 825–836 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sterling, J., Baker, J.

R., McCluskey, A. & Munoz, L. Systematic literature review reveals suboptimal use of chemical probes in cell-based biomedical research. _Nat. Commun._ 14, 3228 (2023). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Li, H. et al. Assigning functionality to cysteines by base editing of cancer dependency genes. _Nat. Chem. Biol._ 19, 1320–1330 (2023). Article  CAS

  PubMed  PubMed Central  Google Scholar  * Chen, Y. et al. Direct mapping of ligandable tyrosines and lysines in cells with chiral sulfonyl fluoride probes. _Nat. Chem._ 15, 1616–1625

(2023). Article  CAS  PubMed  Google Scholar  * Resnick, E. et al. Rapid covalent-probe discovery by electrophile-fragment screening. _J. Am. Chem. Soc._ 141, 8951–8968 (2019). Article  CAS

  PubMed  PubMed Central  Google Scholar  * Dubiella, C. et al. Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo. _Nat. Chem. Biol._ 17, 954–963 (2021). Article

  CAS  PubMed  PubMed Central  Google Scholar  * Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. _Nat. Biotechnol._ 36, 880–887 (2018). Article

  CAS  PubMed  PubMed Central  Google Scholar  * Liu, H. & Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.

_BMC Biotechnol._ 8, 91 (2008). Article  PubMed  PubMed Central  Google Scholar  * Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis.

_Nat. Methods_ 9, 671–675 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool.

_BMC Bioinformatics_ 14, 128 (2013). Article  PubMed  PubMed Central  Google Scholar  * Xie, Z. et al. Gene set knowledge discovery with Enrichr. _Curr. Protoc._ 1, e90 (2021). Article  CAS

  PubMed  PubMed Central  Google Scholar  * Teo, G. C., Polasky, D. A., Yu, F. & Nesvizhskii, A. I. Fast deisotoping algorithm and its implementation in the msfragger search engine. _J.

Proteome Res._ 20, 498–505 (2021). Article  CAS  PubMed  Google Scholar  * Kong, A. T., Leprevost, F. V., Avtonomov, D. M., Mellacheruvu, D. & Nesvizhskii, A. I. MSFragger: ultrafast and

comprehensive peptide identification in mass spectrometry-based proteomics. _Nat. Methods_ 14, 513–520 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Tyanova, S. et al. The

Perseus computational platform for comprehensive analysis of (prote)omics data. _Nat. Methods_ 13, 731–740 (2016). Article  CAS  PubMed  Google Scholar  * Perez-Riverol, Y. et al. The PRIDE

database resources in 2022: a hub for mass spectrometry-based proteomics evidences. _Nucleic Acids Res._ 50, D543–D552 (2022). Article  CAS  PubMed  Google Scholar  Download references

ACKNOWLEDGEMENTS This study was supported by a Beckman Young Investigator Award (to K.M.B.), DOD-Advanced Research Projects Agency (D19AP00041 to K.M.B.), National Institutes of Health (DP2

GM146246-02 to K.M.B.), Packard Fellowship (2020-71388 to K.M.B.) and NIGMS UCLA Chemistry Biology Interface (T32GM136614 to A.R.J.). We thank I. Zohn (Center for Genetic Medicine Research,

Children's National Research and Innovation) for providing the HECTD1 plasmid. We additionally thank all members of the Backus Lab for helpful suggestions. We additionally thank Dr. S.

Neumann (Martin Lab, UCLA) and the UCLA Broad Stem Cell Resource Center Microscopy Core for assistance with microscopy. Figure 1a was made using Biorender.com. AUTHOR INFORMATION Author

notes * These authors contributed equally: Ashley R. Julio, Flowreen Shikwana. AUTHORS AND AFFILIATIONS * Department of Biological Chemistry, David Geffen School of Medicine, UCLA, Los

Angeles, CA, USA Ashley R. Julio, Flowreen Shikwana, Cindy Truong, Nikolas R. Burton, Emil R. Dominguez III, Alexandra C. Turmon, Jian Cao & Keriann M. Backus * Department of Chemistry

and Biochemistry, UCLA, Los Angeles, CA, USA Ashley R. Julio, Flowreen Shikwana, Nikolas R. Burton, Alexandra C. Turmon & Keriann M. Backus * DOE Institute for Genomics and Proteomics,

UCLA, Los Angeles, CA, USA Keriann M. Backus * Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA Keriann M. Backus * Eli and Edythe Broad

Center of Regenerative Medicine and Stem Cell Research, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA Keriann M. Backus Authors * Ashley R. Julio View author publications You

can also search for this author inPubMed Google Scholar * Flowreen Shikwana View author publications You can also search for this author inPubMed Google Scholar * Cindy Truong View author

publications You can also search for this author inPubMed Google Scholar * Nikolas R. Burton View author publications You can also search for this author inPubMed Google Scholar * Emil R.

Dominguez III View author publications You can also search for this author inPubMed Google Scholar * Alexandra C. Turmon View author publications You can also search for this author inPubMed

 Google Scholar * Jian Cao View author publications You can also search for this author inPubMed Google Scholar * Keriann M. Backus View author publications You can also search for this

author inPubMed Google Scholar CONTRIBUTIONS A.R.J., F.S. and K.M.B. conceived of the project. A.R.J., F.S. and K.M.B. designed experiments. A.R.J. and F.S. performed, collected and analyzed

data for all biochemical experiments, including bulk proteomics and protein-directed ABPP, western blot analyses, AP–MS proteomics and cloning of plasmids. F.S. performed, collected and

analyzed data for all isoTOP–ABPP experiments with the help of C.T. and performed all knockdown experiments. A.R.J. performed, collected and analyzed all data for all imaging experiments.

A.C.T. performed, collected and analyzed data for IncuCyte Live-Cell Imaging. N.R.B., E.D. and J.C. performed syntheses of JC19 and all analogs. C.T. developed and implemented software.

A.R.J. and F.S. contributed to the to the preparation and design of figures. A.R.J., F.S. and K.M.B. wrote the manuscript with assistance from all authors. CORRESPONDING AUTHOR

Correspondence to Keriann M. Backus. ETHICS DECLARATIONS COMPETING INTERESTS K.M.B. is a member of the advisory board at Matchpoint Therapeutics. The remaining authors declare no competing

interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Chemical Biology_ thanks Xiaoyu Zhang and the other, anonymous, reviewers for their contribution to the peer review of this work.

ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA

FIG. 1 COMPOUND STRUCTURES. Shown are the structures of all compounds used for the study, ordered in the order of appearance in the text. EXTENDED DATA FIG. 2 JC19 ANALOGS HAVE VARYING

DEGREES OF NSP14 DEPLETION EFFICIENCY. (A) Chemical structures of JC19 analogs. Cysteine-reactive chloroacetamide warhead is colored pink, cysteine-reactive acrylamide warhead is colored

blue and sulfonyl fluoride warhead is colored orange. (B–F) HEK293T cells transiently expressing nsp14-FLAG were treated with the indicated concentrations of JC18 (B), NB92 (C), NB177 (D),

NB179 (E) or JC17 (F) for 0.5 h, and the soluble lysate fraction assayed by immunoblot. (G) HEK293T cells transiently expressing nsp14-FLAG were treated with 100 µm of the indicated

compounds for the indicated times, and the soluble lysate fraction was assayed by immunoblot. All western blot data are representative of 2 independent experiments. Source data EXTENDED DATA

FIG. 3 JC19 INDUCES LINKAGE OF PREDOMINANTLY K48-LINKED POLYUBIQUITIN CHAINS ON NSP14. (A) HEK293T cells transiently expressing nsp14-FLAG were treated with the indicated compounds, lysed

and immunoprecipitated on FLAG resin. (B) HEK293T cells transiently expressing nsp14-FLAG or the indicated ubiquitin construct were treated with DMSO or JC19 (50 µm, 1 h), lysed and

immunoprecipitated on FLAG resin. Immunoblot analysis was used to probe FLAG and ubiquitin expression for the immunoprecipitated fraction. Ubiquitin constructs used include WT (wild-type),

K48R, K63R, K48 (all ubiquitin lysines mutated to arginine except K48) and K63 (all ubiquitin lysines mutated to arginine except K63). (C) Same immunoblot as shown in B, including inputs,

loading control and full-length ubiquitin blot to depict polyubiquitin smears. Polyubiquitylation is primarily due to K48-linked ubiquitin, as indicated by the attenuation (K48R) or

abrogation (K63) of the high-molecular-weight polyubiquitin smear when using ubiquitin constructs containing a mutant K48. (D) ‘Heavy’ SILAC HEK293T cells transiently expressing nsp14-FLAG

were treated with 100 µm JC19 for 1 h (_n_ = 3), while ‘light’ SILAC HEK293T cells transiently expressing nsp14-FLAG were treated with an equal volume of DMSO for 1 h (_n_ = 3). Lysates were

combined, immunoprecipitated on FLAG resin, proteolyzed and subjected to LC–MS/MS analysis. Mean log2(H/L) ratios of detected peptides are depicted, highlighting all nsp14 peptides and

ubiquitinated nsp14 peptides (GlyGly modified). (E) Workflow for AP–MS experiments. All MS data can be found in Supplementary Table 2. Western blot data are representative of 2 independent

experiments. Source data EXTENDED DATA FIG. 4 COMMON CELL STRESS-SENSING PATHWAYS ARE NOT RESPONSIBLE FOR NSP14 DEPLETION. (A) HEK293T cells transiently expressing nsp14-FLAG were treated

with the indicated concentrations of JC19 (1 h). Immunoblot analysis was used to visualize abundance of nsp14 and induction of NRF2 expression in the soluble lysate fraction (0.3% CHAPS in

PBS) for each condition. (B) HEK293T cells transiently expressing nsp14-FLAG were pretreated with either DMSO, tunicamycin (12 µg/mL, 8 h), thapsigargin (2 µm, 8 h) or rapamycin (1 µm, 8 h),

and then either treated with DMSO or JC19 (50 µm, 1 h). Immunoblot analysis was used to visualize abundance of nsp14 and induction of UPR markers in the soluble lysate fraction (0.3% CHAPS

in PBS) for each condition. (C) HEK293T cells were treated with the indicated compounds, and immunoblot analysis was used to visualize induction of UPR markers (lysed in RIPA). (D–F) HEK293T

cells were treated with vehicle DMSO, thapsigargin (2 µm, 15 h) or AA147 (10 µm, 15 h; _n_ = 3 per group; (D) or DMSO, 10 µm JC19 for 1 h or 10 µm JC19 for 15 h (_n_ = 3 per group; (E) or

DMSO, 50 µm JC19 for 1 h or 50 µm JC19 for 15 h (_n_ = 3 per group; (F) and lysed in RIPA lysis buffer. Bulk proteomics sample preparation and label-free quantification proteomics were used

to measure intensities of proteins and p-values generated for each treatment compared to vehicle using a Student’s unpaired t-test. −log10(p-values) were plotted and significance of BiP

(HSPA5) and PDIA4 highlighted. All MS data can be found in Supplementary Table 2. Panel A is representative of one independent measurement, and panels B and C are representative of three

independent measurements. Source data EXTENDED DATA FIG. 5 PDIA6-HIS PULLDOWN. (A) HEK293T cells transiently expressing PDIA6-His protein were treated with DMSO, NB92 or JC19 for 1 h at 50 

µm. Cells were lysed, and each condition was split into 2 tubes, one treated with TCEP and one without TCEP. Samples were then incubated with Ni-NTA resin, washed, eluted and prepared for

LC–MS/MS analysis. Proteins significantly enriched by JC19 in both the +TCEP and −TCEP conditions were identified to be true cross-linked proteins to PDIA6. Proteins enriched by JC19 only in

the −TCEP condition but not +TCEP condition were identified to be linked to PDIA6 by disulfide bond. (B) HEK293T cells transiently expressing PDIA6-His8x were affinity purified under

denaturing conditions either in the absence (black border labels) or presence of TCEP reductant (green border labels). Volcano plot displays comparison between enriched proteins for groups

treated with DMSO versus NB92 (50 µm, 1 h) with black colored dots for proteins showing sensitivity to TCEP (_n_ = 3 for each group). An unpaired Student’s t-test was performed to calculate

p-values. All MS data can be found in Supplementary Table 3. EXTENDED DATA FIG. 6 HIGH-RATIO CYSTEINES AS IDENTIFIED BY ISOTOP–ABPP BELONG TO PROTEINS SPANNING VARIOUS SUBCELLULAR

COMPARTMENTS. Heatmap depicting unlogged MS1 isoTOP–ABPP cysteine ratios for high coverage cysteines belonging to proteins with multiple cysteines identified. Cell compartment annotations

from UniProt have been provided for each cysteine identifier. For generation of data, each compound was used in triplicate (n = 3) at 100 µm for 1 h (exception: 4 h treatment for EN450) in

nsp14-expressing HEK293T cells. MS data can be found in Supplementary Table 4. EXTENDED DATA FIG. 7 DELINEATION OF AGGREGATION AND DEGRADATION PROPERTIES OF JC19, EN450 AND NB001. (A)

HEK293T cells transiently expressing nsp14-FLAG were treated with DMSO, a low dose of JC19 (25 µm for 0.5 h) or a high dose of JC19 (100 µm for 1 h) and subjected to immunoblot analysis.

Cells were lysed in 0.3% CHAPS in PBS to generate the soluble lysate, and after clearance by centrifugation, the insoluble debris was solubilized in 8 M urea in PBS to generate ‘insoluble’

lysate. (B) HEK293T cells transiently expressing nsp14-FLAG were treated with the indicated concentrations of JC19 or NB001 for 0.5 h or EN450 for 3 h, and the insoluble lysate was analyzed

by immunoblot. (C) Quantification of nsp14 intensity from replicate western blots as in B. Data are presented as mean ± s.d. (_n_ = 3 per data point). D,E, Nsp14-expressing HEK293T cells

were treated with the indicated concentrations of JC19 for 0.5 h (D) or EN450 for 3 h (E), and were then separated into 3 fractions: ‘soluble’ fraction (cells lysed in 0.3% CHAPS and cleared

by centrifugation), ‘insoluble’ fraction (insoluble debris from CHAPS lysis re-solubilized using 8 M urea) and ‘total’ fraction (cells lysed in 8 M urea with 3× freeze/thaw). (F) HEK293T

cells transiently expressing nsp14-FLAG were treated with the indicated concentrations of JC19 or NB001 for 0.5 h or EN450 for 3 h, and the soluble and insoluble lysates were analyzed by

immunoblot. (G) Quantification of nsp14 intensity from replicate western blots as in F. Data are presented as mean ± s.d. (_n_ = 3 per data point). All western blot data is representative of

three independent measurements. Source data EXTENDED DATA FIG. 8 VARIOUS CYSTEINE-REACTIVE ELECTROPHILES INDUCE STRESS GRANULE FORMATION. (A) U2OS cells stably expressing V5-tagged G3BP1

were treated with DMSO, RA190 (10 µm, 1 h), sulforaphane (50 µm, 1 h), bardoxolone (10 µm, 1 h), ibrutinib (10 µm, 14 h), low afatinib (100 nM for 14 h), high afatinib (10 µm, 1 h) or

auranofin (10 µm, 1 h). Cells were then fixed, permeabilized and subjected to immunofluorescence microscopy. (B) U2OS cells stably expressing V5-tagged G3BP1 were treated with the indicated

concentration of afatinib for 4 h. Cells were then fixed, permeabilized and subjected to immunofluorescence microscopy. (C) Immunofluorescence microscopy of G3BP1 and RANBP2 in response to

DMSO, JC19 (100 µm, 1 h) and EN450 (100 µm, 1 h). (D) Immunofluorescence microscopy of G3BP1 and PSMB2 in response to DMSO and JC19 (100 µm, 1 h). Images were acquired on an LSM880 confocal

microscope at ×63 objective and 2× manual zoom. All scale bars = 10 µm. All images are representative of two independent measurements. EXTENDED DATA FIG. 9 GENETIC KNOCKDOWN OF PDIA3 AND

PDIA6 LEAD TO INCREASES IN AGGREGATED PROTEINS (AGGRESOMES). (A) Immunofluorescence microscopy of HEK293T cells transfected with either a non-targeting control (NTC) siRNA for 48 h, NTC

siRNA for 48 h and treated with MG132 (10 µm, 8 h), PDIA3 siRNA for 48 h or PDIA6 siRNA for 48 h, using p62 as a marker for aggresomes/aggregated proteins. Images were acquired on an LSM880

confocal microscope at ×63 objective and 2× manual zoom. All scale bars = 10 µm. (B) Immunoblot analysis PDIA3 and PDIA6 knockdown efficiency for the cells used in A and Fig. 6e. All data

are representative of three independent measurements. Source data EXTENDED DATA FIG. 10 RECOMMENDATIONS FOR BEST PRACTICES. Suggested steps to facilitate delineation of on- versus off-target

activities of covalent fragments. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–30, supporting data of Supplementary Figs. 2–11, 14–16, 21, 23–26 (uncropped and

unprocessed western blots), Supplementary Tables 6–10 and Supplementary Note. REPORTING SUMMARY SUPPLEMENTARY TABLE 1 MS datasets corresponding to Fig. 1. SUPPLEMENTARY TABLE 2 MS datasets

corresponding to Fig. 3. SUPPLEMENTARY TABLE 3 MS datasets corresponding to Fig. 4. SUPPLEMENTARY TABLE 4 MS datasets corresponding to Fig. 5. SUPPLEMENTARY TABLE 5 MS datasets corresponding

to Fig. 6. SUPPLEMENTARY TABLE 11 Annotation of all files uploaded to the PRIDE. SUPPLEMENTARY DATA 1 Raw data corresponding to data shown in Supplementary Fig. 2b. SUPPLEMENTARY DATA 2 Raw

data corresponding to data shown in Supplementary Fig. 11b. SOURCE DATA SOURCE DATA FIG. 1 Uncropped blots. SOURCE DATA FIG. 2 Uncropped blots. SOURCE DATA FIG. 2 Raw data corresponding to

data shown in Fig. 2e. SOURCE DATA FIG. 3 Uncropped blots. SOURCE DATA FIG. 4 Uncropped blots. SOURCE DATA FIG. 4 Raw data corresponding to data shown in Fig. 4k. SOURCE DATA FIG. 5

Uncropped blots. SOURCE DATA FIG. 5 Raw data corresponding to data shown in Fig. 5h. SOURCE DATA FIG. 6 Raw data corresponding to data shown in Fig. 6c. SOURCE DATA EXTENDED DATA FIG. 2

Uncropped blots. SOURCE DATA EXTENDED DATA FIG. 3 Uncropped blots. SOURCE DATA EXTENDED DATA FIG. 4 Uncropped blots. SOURCE DATA EXTENDED DATA FIG. 7 Source data. SOURCE DATA EXTENDED DATA

FIG. 7 Uncropped blots. SOURCE DATA EXTENDED DATA FIG. 9 Uncropped blots. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to

this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the

terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Julio, A.R., Shikwana, F., Truong, C. _et al._ Delineating

cysteine-reactive compound modulation of cellular proteostasis processes. _Nat Chem Biol_ 21, 693–705 (2025). https://doi.org/10.1038/s41589-024-01760-9 Download citation * Received: 16

November 2023 * Accepted: 23 September 2024 * Published: 24 October 2024 * Issue Date: May 2025 * DOI: https://doi.org/10.1038/s41589-024-01760-9 SHARE THIS ARTICLE Anyone you share the

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