Nucleophilic covalent ligand discovery for the cysteine redoxome.

With an eye toward expanding chemistries used for covalent ligand discovery, we elaborated an umpolung strategy that exploits the 'polarity reversal' of sulfur when cysteine is oxidized to sulfenic acid, a widespread post-translational modification, for selective bioconjugation with C-nucleophiles. Here we present a global map of a human sulfenome that is susceptible to covalent modification by members of a nucleophilic fragment library. More than 500 liganded sulfenic acids were identified on proteins across diverse functional classes, and, of these, more than 80% were not targeted by electrophilic fragment analogs. We further show that members of our nucleophilic fragment library can impair functional protein-protein interactions involved in nuclear oncoprotein transport and DNA damage repair. Our findings reveal a vast expanse of ligandable sulfenic acids in the human proteome and highlight the utility of nucleophilic small molecules in the fragment-based covalent ligand discovery pipeline, presaging further opportunities using non-traditional chemistries for targeting proteins.

Sulfenylation links oxidative stress to protein disulfide isomerase oxidase activity and thrombus formation.

BACKGROUND: Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines. OBJECTIVES: We hypothesized that PDI is sulfenylated during oxidative stress, contributing to the prothrombotic potential of PDI. METHODS: Biochemical and enzymatic assays using purified proteins, platelet and endothelial cell assays, and in vivo murine thrombosis studies were used to evaluate the role of oxidative stress in PDI sulfenylation and prothrombotic activity. RESULTS: PDI exposure to oxidants resulted in the loss of PDI reductase activity and simultaneously promoted sulfenylated PDI generation. Following exposure to oxidants, sulfenylated PDI spontaneously converted to disulfided PDI. PDI oxidized in this manner was able to transfer disulfides to protein substrates. Inhibition of sulfenylation impaired disulfide formation by oxidants, indicating that sulfenylation is an intermediate during PDI oxidation. Agonist-induced activation of platelets and endothelium resulted in the release of sulfenylated PDI. PDI was also sulfenylated by oxidized low-density lipoprotein (oxLDL). In an in vivo model of thrombus formation, oxLDL markedly promoted platelet accumulation following an arteriolar injury. PDI oxidoreductase inhibition blocked oxLDL-mediated augmentation of thrombosis. CONCLUSION: PDI sulfenylation is a critical posttranslational modification that is an intermediate during disulfide PDI formation in the setting of oxidative stress. Oxidants generated by vascular cells during activation promote PDI sulfenylation, and interference with PDI during oxidative stress impairs thrombus formation.

Chaperone-directed ribosome repair after oxidative damage.

Because of the central role ribosomes play for protein translation and ribosome-mediated mRNA and protein quality control (RQC), the ribosome pool is surveyed and dysfunctional ribosomes degraded both during assembly, as well as the functional cycle. Oxidative stress downregulates translation and damages mRNAs and ribosomal proteins (RPs). Although damaged mRNAs are detected and degraded via RQC, how cells mitigate damage to RPs is not known. Here, we show that cysteines in Rps26 and Rpl10 are readily oxidized, rendering the proteins non-functional. Oxidized Rps26 and Rpl10 are released from ribosomes by their chaperones, Tsr2 and Sqt1, and the damaged ribosomes are subsequently repaired with newly made proteins. Ablation of this pathway impairs growth, which is exacerbated under oxidative stress. These findings reveal an unanticipated mechanism for chaperone-mediated ribosome repair, augment our understanding of ribosome quality control, and explain previous observations of protein exchange in ribosomes from dendrites, with broad implications for aging and health.

Reaction-based fluorogenic probes for detecting protein cysteine oxidation in living cells.

'Turn-on' fluorescence probes for detecting H2O2 in cells are established, but equivalent tools to monitor the products of its reaction with protein cysteines have not been reported. Here we describe fluorogenic probes for detecting sulfenic acid, a redox modification inextricably linked to H2O2 signaling and oxidative stress. The reagents exhibit excellent cell permeability, rapid reactivity, and high selectivity with minimal cytotoxicity. We develop a high-throughput assay for measuring S-sulfenation in cells and use it to screen a curated kinase inhibitor library. We reveal a positive association between S-sulfenation and inhibition of TK, AGC, and CMGC kinase group members including GSK3, a promising target for neurological disorders. Proteomic mapping of GSK3 inhibitor-treated cells shows that S-sulfenation sites localize to the regulatory cysteines of antioxidant enzymes. Our studies highlight the ability of kinase inhibitors to modulate the cysteine sulfenome and should find broad application in the rapidly growing field of redox medicine.

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein.

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.

Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting.

Triphenylphosphonium ylides, known as Wittig reagents, are one of the most commonly used tools in synthetic chemistry. Despite their considerable versatility, Wittig reagents have not yet been explored for their utility in biological applications. Here we introduce a chemoselective ligation reaction that harnesses the reactivity of Wittig reagents and the unique chemical properties of sulfenic acid, a pivotal post-translational cysteine modification in redox biology. The reaction, which generates a covalent bond between the ylide nucleophilic α-carbon and electrophilic γ-sulfur, is highly selective, rapid and affords robust labelling under a range of biocompatible reaction conditions, which includes in living cells. We highlight the broad utility of this conjugation method to enable site-specific proteome-wide stoichiometry analysis of S-sulfenylation and to visualize redox-dependent changes in mitochondrial cysteine oxidation and redox-triggered triphenylphosphonium generation for the controlled delivery of small molecules to mitochondria.

Parallel evaluation of nucleophilic and electrophilic chemical probes for sulfenic acid: Reactivity, selectivity and biocompatibility.

Cysteine sulfenic acids (Cys-SOH) are pivotal modifications in thiol-based redox signaling and central intermediates en route to disulfide and sulfinic acid states. A core mission in our lab is to develop bioorthogonal chemical tools with the potential to answer mechanistic questions involving cysteine oxidation. Our group, among others, has contributed to the development of nucleophilic chemical probes for detecting sulfenic acids in living cells. Recently, another class of Cys-SOH probes based on strained alkene and alkyne electrophiles has emerged. However, the use of different models of sulfenic acid and methodologies, has confounded clear comparison of these probes with respect to chemical reactivity, kinetics, and selectivity. Here, we perform a parallel evaluation of nucleophilic and electrophilic chemical probes for Cys-SOH. Among the key findings, we demonstrate that a probe for Cys-SOH based on the norbornene scaffold does not react with any of the validated sulfenic acid models in this study. Furthermore, we show that purported cross-reactivity of dimedone-like probes with electrophiles, like aldehydes and cyclic sulfenamides, is a not meaningful in a biological setting. In summary, nucleophilic probes remain the most viable tools for bioorthogonal detection of Cys-SOH.

Crystal Structure of the [4Fe-4S] Cluster-Containing Adenosine-5'-phosphosulfate Reductase from Mycobacterium tuberculosis.

Tuberculosis (TB) is the deadliest infectious disease in the world. In Mycobacterium tuberculosis, the first committed step in sulfate assimilation is the reductive cleavage of adenosine-5'-phosphosulfate (APS) to form adenosine-5'-phosphate (AMP) and sulfite by the enzyme APS reductase (APSR). The vital role of APSR in the production of essential reduced-sulfur-containing metabolites and the absence of a homologue enzyme in humans makes APSR a potential target for therapeutic interventions. Here, we present the crystal structure of the [4Fe-4S] cluster-containing APSR from M. tuberculosis (MtbAPSR) and compare it to previously determined structures of sulfonucleotide reductases. We further present MtbAPSR structures with substrate APS and product AMP bound in the active site. Our structures at a 3.1 Å resolution show high structural similarity to other sulfonucleotide reductases and reveal that APS and AMP have similar binding modes. These studies provide structural data for structure-based drug design aimed to combat TB.

Endogenous SO2-dependent Smad3 redox modification controls vascular remodeling.

Sulfur dioxide (SO2) has emerged as a physiological relevant signaling molecule that plays a prominent role in regulating vascular functions. However, molecular mechanisms whereby SO2 influences its upper-stream targets have been elusive. Here we show that SO2 may mediate conversion of hydrogen peroxide (H2O2) to a more potent oxidant, peroxymonosulfite, providing a pathway for activation of H2O2 to convert the thiol group of protein cysteine residues to a sulfenic acid group, aka cysteine sulfenylation. By using site-centric chemoproteomics, we quantified >1000 sulfenylation events in vascular smooth muscle cells in response to exogenous SO2. Notably, ~42% of these sulfenylated cysteines are dynamically regulated by SO2, among which is cysteine-64 of Smad3 (Mothers against decapentaplegic homolog 3), a key transcriptional modulator of transforming growth factor ß signaling. Sulfenylation of Smad3 at cysteine-64 inhibits its DNA binding activity, while mutation of this site attenuates the protective effects of SO2 on angiotensin II-induced vascular remodeling and hypertension. Taken together, our findings highlight the important role of SO2 in vascular pathophysiology through a redox-dependent mechanism.

Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans.

Post-translational changes in the redox state of cysteine residues can rapidly and reversibly alter protein functions, thereby modulating biological processes. The nematode C. elegans is an ideal model organism for studying cysteine-mediated redox signaling at a network level. Here we present a comprehensive, quantitative, and site-specific profile of the intrinsic reactivity of the cysteinome in wild-type C. elegans. We also describe a global characterization of the C. elegans redoxome in which we measured changes in three major cysteine redox forms after H2O2 treatment. Our data revealed redox-sensitive events in translation, growth signaling, and stress response pathways, and identified redox-regulated cysteines that are important for signaling through the p38 MAP kinase (MAPK) pathway. Our in-depth proteomic dataset provides a molecular basis for understanding redox signaling in vivo, and will serve as a valuable and rich resource for the field of redox biology.

Cysteine sulfenylation by CD36 signaling promotes arterial thrombosis in dyslipidemia.

Arterial thrombosis in the setting of dyslipidemia promotes clinically significant events, including myocardial infarction and stroke. Oxidized lipids in low-density lipoproteins (oxLDL) are a risk factor for athero-thrombosis and are recognized by platelet scavenger receptor CD36. oxLDL binding to CD36 promotes platelet activation and thrombosis by promoting generation of reactive oxygen species. The downstream signaling events initiated by reactive oxygen species in this setting are poorly understood. In this study, we report that CD36 signaling promotes hydrogen peroxide flux in platelets. Using carbon nucleophiles that selectively and covalently modify cysteine sulfenic acids, we found that hydrogen peroxide generated through CD36 signaling promotes cysteine sulfenylation of platelet proteins. Specifically, cysteines were sulfenylated on Src family kinases, which are signaling transducers that are recruited to CD36 upon recognition of its ligands. Cysteine sulfenylation promoted activation of Src family kinases and was prevented by using a blocking antibody to CD36 or by enzymatic degradation of hydrogen peroxide. CD36-mediated platelet aggregation and procoagulant phosphatidylserine externalization were inhibited in a concentration-dependent manner by a panel of sulfenic acid-selective carbon nucleophiles. At the same concentrations, these probes did not inhibit platelet aggregation induced by the purinergic receptor agonist adenosine diphosphate or the collagen receptor glycoprotein VI agonist collagen-related peptide. Selective modification of cysteine sulfenylation in vivo with a benzothiazine-based nucleophile rescued the enhanced arterial thrombosis seen in dyslipidemic mice back to control levels. These findings suggest that CD36 signaling generates hydrogen peroxide to oxidize cysteines within platelet proteins, including Src family kinases, and lowers the threshold for platelet activation in dyslipidemia.

Activity-Based Sensing for Site-Specific Proteomic Analysis of Cysteine Oxidation.

Oxidative post-translational modifications (OxiPTMs) of cysteine residues are the molecular foundation of thiol-based redox regulation that modulates physiological events such as cell proliferation, differentiation, and migration and, when dysregulated, can lead to biomolecule damage and cell death. Common OxiPTMs of cysteine thiols (-SH) include reversible modifications such as S-sulfenylation (-SOH), S-glutathionylation (-SSG), disulfide formation (-SSR), S-nitrosylation (-SNO), and S-sulfhydration (-SSH) as well as more biologically stable modifications like S-sulfinylation (-SO2H) and S-sulfonylation (-SO3H). In the past decade, our laboratory has developed first-in-class chemistry-based tools and proteomic methods to advance the field of thiol-based redox biology and oxidative stress. In this Account, we take the reader through the historical aspects of probe development and application in our laboratory, highlighting key advances in our understanding of sulfur chemistry, in the test tube and in living systems. Offering superior resolution, throughput, accuracy, and reproducibility, mass spectrometry (MS)-based proteomics coupled to chemoselective "activity-based" small-molecule probes is the most rigorous technique for global mapping of cysteine OxiPTMs. Herein, we describe the evolution of this field from indirect detection to state-of-the-art site-centric quantitative chemoproteomic approaches that enable mapping of physiological and pathological changes in cysteine oxidation. These methods enable protein and site-level identification, mechanistic studies, mapping fold-changes, and modification stoichiometry. In particular, this Account focuses on activity-based methods for profiling S-sulfenylation, S-sulfinylation, and S-sulfhydration with an eye toward new reactions and methodologies developed in our group as well as their applications that have shed new light on fundamental processes of redox biology. Among several classes of sulfenic acid probes, dimedone-based C-nucleophiles possess superior chemical selectivity and compatibility with tandem MS. Cell-permeable dimedone derivatives with a bioconjugation handle are capable of detecting of S-sulfenylation in living cells. In-depth screening of a C-nucleophile library has yielded several entities with significantly enhanced reactivity over dimedone while maintaining selectivity, and reversible linear C-nucleophiles that enable controlled target release. C-Nucleophiles have also been implemented in tag-switch methods to detect S-sulfhydration. Most recently, activity-based detection of protein S-sulfinylation with electrophilic nitrogen species (ENS), such as C-nitroso compounds and electron deficient diazines, offers significant advantages in simplicity-of-use and target specificity compared to label-free methods. When feasible, the rich information provided by site-centric quantitative proteomics should not be tainted by oxidation artifacts from cell lysis. Therefore, chemoselective probes that function in a native environment with low cytotoxicity, good cell-permeability, and competitive kinetics are desired in modern redox chemoproteomics approaches. As our understanding of sulfur chemistry and redox signaling evolves, newly discovered cysteine OxiPTMs in microorganisms, plants, cells, tissues, and disease models should innovatively promote mechanistic and therapeutic research.

Selective Persulfide Detection Reveals Evolutionarily Conserved Antiaging Effects of S-Sulfhydration.

Life on Earth emerged in a hydrogen sulfide (H2S)-rich environment eons ago and with it protein persulfidation mediated by H2S evolved as a signaling mechanism. Protein persulfidation (S-sulfhydration) is a post-translational modification of reactive cysteine residues, which modulate protein structure and/or function. Persulfides are difficult to label and study due to their reactivity and similarity with cysteine. Here, we report a facile strategy for chemoselective persulfide bioconjugation using dimedone-based probes, to achieve highly selective, rapid, and robust persulfide labeling in biological samples with broad utility. Using this method, we show persulfidation is an evolutionarily conserved modification and waves of persulfidation are employed by cells to resolve sulfenylation and prevent irreversible cysteine overoxidation preserving protein function. We report an age-associated decline in persulfidation that is conserved across evolutionary boundaries. Accordingly, dietary or pharmacological interventions to increase persulfidation associate with increased longevity and improved capacity to cope with stress stimuli.

Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites.

Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H2O2 stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.

Proteome-Wide Analysis of Cysteine S-Sulfenylation Using a Benzothiazine-Based Probe.

Oxidation of a protein cysteinyl thiol (Cys-SH) to S-sulfenic acid (Cys-SOH) by a reactive oxygen species (e.g., hydrogen peroxide), which is termed protein S-sulfenylation, is a reversible post-translational modification that plays a crucial role in redox regulation of protein function in various biological processes. Due to its intrinsically labile nature, protein S-sulfenylation cannot be directly detected or analyzed. Chemoselective probing has been the method of choice for analyzing S-sulfenylated proteins either in vitro or in situ, as it allows stabilization and direct detection of this transient oxidative intermediate. However, it remains challenging to globally pinpoint the specific S-sulfenylated cysteine sites on complex proteomes and to quantify their dynamic changes upon oxidative stress. This unit describes how a benzothiazine-based chemoselective probe called BTD and mass spectrometry based chemoproteomics can be used to globally and site-specifically identify and quantify protein S-sulfenylation. © 2018 by John Wiley & Sons, Inc.

Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice.

The gain-of-function MUC5B promoter variant rs35705950 is the dominant risk factor for developing idiopathic pulmonary fibrosis (IPF). Here we show in humans that MUC5B, a mucin thought to be restricted to conducting airways, is co-expressed with surfactant protein C (SFTPC) in type 2 alveolar epithelia and in epithelial cells lining honeycomb cysts, indicating that cell types involved in lung fibrosis in distal airspace express MUC5B. In mice, we demonstrate that Muc5b concentration in bronchoalveolar epithelia is related to impaired mucociliary clearance (MCC) and to the extent and persistence of bleomycin-induced lung fibrosis. We also establish the ability of the mucolytic agent P-2119 to restore MCC and to suppress bleomycin-induced lung fibrosis in the setting of Muc5b overexpression. Our findings suggest that mucociliary dysfunction might play a causative role in bleomycin-induced pulmonary fibrosis in mice overexpressing Muc5b, and that MUC5B in distal airspaces is a potential therapeutic target in humans with IPF.

Chemical proteomics reveals new targets of cysteine sulfinic acid reductase.

Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid modification (S-sulfenylation) and the S-sulfinylation dynamic response to oxidative stress, which is indicative of different roles for these OxiPTMs in redox regulation. We also identified >55 heretofore-unknown protein substrates of the cysteine sulfinic acid reductase sulfiredoxin, extending its function well beyond those of 2-cysteine peroxiredoxins (2-Cys PRDX1-4) and offering new insights into the role of this unique oxidoreductase as a central mediator of reactive oxygen species-associated diseases, particularly cancer. DiaAlk therefore provides a novel tool to profile S-sulfinylated proteins and study their regulatory mechanisms in cells.

Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles.

Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this process is the identification of signaling pathways (or receptors) specific to (or overexpressed in) disease cells. In this context, fragment-based ligand discovery (FBLD) has significantly expanded our view of the ligandable proteome and affords tool compounds for biological inquiry. To date, such covalent ligand discovery has almost exclusively employed cysteine-reactive small-molecule fragments. However, functional cysteine residues in proteins are often redox-sensitive and can undergo oxidation in cells. Such reactions are particularly relevant in diseases, like cancer, which are linked to excessive production of reactive oxygen species. Once oxidized, the sulfur atom of cysteine is much less reactive toward electrophilic groups used in the traditional FBLD paradigm. To address this limitation, we recently developed a novel library of diverse carbon-based nucleophile fragments that react selectively with cysteine sulfenic acid formed in proteins via oxidation or hydrolysis reactions. Here, we report analysis of sulfenic acid-reactive C-nucleophile fragments screened against a colon cancer cell proteome. Covalent ligands were identified for >1280 S-sulfenylated cysteines present in "druggable" proteins and orphan targets, revealing disparate reactivity profiles and target preferences. Among the unique ligand-protein interactions identified was that of a pyrrolidinedione nucleophile that reacted preferentially with protein tyrosine phosphatases. Fragment-based covalent ligand discovery with C-nucleophiles affords an expansive snapshot of the ligandable "redoxome" with significant implications for covalent inhibitor pharmacology and also affords new chemical tools to investigate redox-regulation of protein function.