Reactive Cysteine Residues Research Articles

Stepwise oxidations play key roles in the structural and functional regulations of DJ-1.

DJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. Human DJ-1 has three cysteine residues (Cys46, Cys53 and Cys106). We found that, in addition to Cys106, Cys46 is the most reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe (Ctag) that selectively reacts with redox-sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with adjacent resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. Mutants (C46A and C53A), not forming Cys46-Cys53 disulfide cross-linking, increased oxidation of Cys106 to sulfinic and sulfonic acids. Furthermore, we found that DJ-1 C46A mutant has distorted unstable structure identified by biochemical assay and employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike WT DJ-1. These findings suggest that all three Cys residues including Cys46-Cys53 disulfide cross-linking are required for maintaining the structural integrity, the regulation process and cellular function as an antioxidant protein. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.

Searching Geometric Patterns in Protein Binding Sites and Their Application to Data Mining in Protein Kinase Structures.

The ever-growing number of protein-ligand complex structures can give fundamental insights into protein functions and protein-ligand interactions, especially in the field of protein kinase research. The number of tools to mine this data for individually defined structural motifs is restricted due to the challenging task of developing efficient index structures for 3D data in relational databases. Herein we present GeoMine, a database system with web front-end mining of more than 900 000 binding sites. It enables database searches for geometric (interaction) patterns in protein-ligand interfaces by, for example, textual, numerical, substructure, similarity, and 3D searches. GeoMine processes reasonably selective user-defined queries within minutes. We demonstrate its usability for advancing protein kinase research with a special emphasis on unusual interactions, their use in designing selective kinase inhibitors, and the analysis of reactive cysteine residues that are amenable to covalent kinase inhibitors. GeoMine is freely available as part of our modeling support server at https://proteins.plus.

Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit.

In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate.

Relevance of peroxiredoxins in pathogenic microorganisms.

The oxidative and nitrosative responses generated by animals and plants are important defenses against infection and establishment of pathogenic microorganisms such as bacteria, fungi, and protozoa. Among distinct oxidant species, hydroperoxides are a group of chemically diverse compounds that comprise small hydrophilic molecules, such as hydrogen peroxide and peroxynitrite, and bulky hydrophobic species, such as organic hydroperoxides. Peroxiredoxins (Prx) are ubiquitous enzymes that use a highly reactive cysteine residue to decompose hydroperoxides and can also perform other functions, like molecular chaperone and phospholipase activities, contributing to microbial protection against the host defenses. Prx are present in distinct cell compartments and, in some cases, they can be secreted to the extracellular environment. Despite their high abundance, Prx expression can be further increased in response to oxidative stress promoted by host defense systems, by treatment with hydroperoxides or by antibiotics. In consequence, some isoforms have been described as virulence factors, highlighting their importance in pathogenesis. Prx are very diverse and are classified into six different classes (Prx1-AhpC, BCP-PrxQ, Tpx, Prx5, Prx6, and AhpE) based on structural and biochemical features. Some groups are absent in hosts, while others present structural peculiarities that differentiate them from the host's isoforms. In this context, the intrinsic characteristics of these enzymes may aid the development of new drugs to combat pathogenic microorganisms. Additionally, since some isoforms are also found in the extracellular environment, Prx emerge as attractive targets for the production of diagnostic tests and vaccines. KEY POINTS: • Peroxiredoxins are front-line defenses against host oxidative and nitrosative stress. • Functional and structural peculiarities differ pathogen and host enzymes. • Peroxiredoxins are potential targets to microbicidal drugs.

Proteomic Approaches to Study Cysteine Oxidation: Applications in Neurodegenerative Diseases.

Oxidative stress appears to be a key feature of many neurodegenerative diseases either as a cause or consequence of disease. A range of molecules are subject to oxidation, but in particular, proteins are an important target and measure of oxidative stress. Proteins are subject to a range of oxidative modifications at reactive cysteine residues, and depending on the level of oxidative stress, these modifications may be reversible or irreversible. A range of experimental approaches has been developed to characterize cysteine oxidation of proteins. In particular, mass spectrometry-based proteomic methods have emerged as a powerful means to identify and quantify cysteine oxidation sites on a proteome scale; however, their application to study neurodegenerative diseases is limited to date. Here we provide a guide to these approaches and highlight the under-exploited utility of these methods to measure oxidative stress in neurodegenerative diseases for biomarker discovery, target engagement and to understand disease mechanisms.

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB2 by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB2 in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB2.

S-desulfurization: A different covalent modification mechanism from persulfidation by GSH

Post-translational transformation of cysteine residues to persulfides, known as protein S-sulfhydration or persulfidation, is a beneficial H2S signaling mechanism. In this paper, we found that GSH is bound to active cysteine sites of protein by S-desulfurization, which is a new covalent modification mechanism of protein, thus regulating catalytic activity. Here, we provide direct evidence that GSH modifies the reactive cysteine residues of four enzymes (alliinase/D-LDH/ADH/G6PD) and generates protein-SG or protein-SSG derivatives by S-desulfurization. S-desulfurization, α-carbon nucleophilic substitution or thiol-disulfide exchange occurs and H2S is released as a by-product. S-desulfurization is the opposite of persulfidation in terms of H2S production/consumption and enzyme inhibition/mitigation. Here, we elucidated the GSH mechanisms and H2S mechanisms in the enzyme-metabolite system and the beneficial roles of persulfidation and S-desulfurization. These theoretical findings are now shedding light on understanding GSH and H2S molecular functions and providing new theoretical basis for them in cell signaling pathways.

Chloride-dependent conformational changes in the GlyT1 glycine transporter

The human GlyT1 glycine transporter requires chloride for its function. However, the mechanism by which Cl- exerts its influence is unknown. To examine the role that Cl- plays in the transport cycle, we measured the effect of Cl- on both glycine binding and conformational changes. The ability of glycine to displace the high-affinity radioligand [3H]CHIBA-3007 required Na+ and was potentiated over 1,000-fold by Cl- We generated GlyT1b mutants containing reactive cysteine residues in either the extracellular or cytoplasmic permeation pathways and measured changes in the reactivity of those cysteine residues as indicators of conformational changes in response to ions and substrate. Na+ increased accessibility in the extracellular pathway and decreased it in the cytoplasmic pathway, consistent with stabilizing an outward-open conformation as observed in other members of this transporter family. In the presence of Na+, both glycine and Cl- independently shifted the conformation of GlyT1b toward an outward-closed conformation. Together, Na+, glycine, and Cl- stabilized an inward-open conformation of GlyT1b. We then examined whether Cl- acts by interacting with a conserved glutamine to allow formation of an ion pair that stabilizes the closed state of the extracellular pathway. Molecular dynamics simulations of a GlyT1 homolog indicated that this ion pair is formed more frequently as that pathway closes. Mutation of the glutamine blocked the effect of Cl-, and substituting it with glutamate or lysine resulted in outward- or inward-facing transporter conformations, respectively. These results provide an unexpected insight into the role of Cl- in this family of transporters.

Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries.

Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS)G12C and Bruton's tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.

Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury.

Background and Purpose: Kelch ECH-associating protein 1 (Keap1) is a crucial chaperonin for E3 ubiquitin ligases. Modification of the key reactive cysteine residues in Keap1 affects the interaction between Keap1 and its substrate nuclear factor erythroid 2-related factor 2 (Nrf2), subsequently regulating oxidative stress and NLPR3 inflammasome activation, which are important factors for myocardial ischemia-reperfusion injury (MI/RI). Pubescenoside A (PBA), an active compound from Ilex pubescens, has antithrombotic and anti-inflammatory effects. However, the effect of PBA on MI/RI is still unknown. In the present study, we aimed to determine whether PBA can protect the heart against MI/RI and clarify the direct target and the underlying mechanism of PBA.Methods: The left anterior descending artery (LAD) ligation-induced MI/RI mice model or oxygen and glucose deprivation/reperfusion (OGD/R) were used to evaluate the cardioprotective effect of PBA. Pull-down assays, co-immunoprecipitation (Co-IP) assays, LC/MS/MS, isothermal calorimetry (ITC) experiments and covalent docking were used to identify the target of PBA.Results: PBA protected cardiomyocytes against OGD/R in vitro and LAD-induced MI/RI in vivo. PBA suppressed NLRP3 inflammation activation and induced the Nrf2 signaling pathway. Interestingly, PBA targeted Keap1 by selectively covalently binding to conserved cysteine residues, cysteine 77 (Cys77) in the BTB domain and cysteine 434 (Cys434) in the Kelch domain of Keap1, subsequently inhibiting ubiquitination of Nrf2 and activating antioxidant enzymes. Additionally, the cysteines of Keap1 has different degree of activation by PBA as follows: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which further elucidates the cysteine sensitivity of Keap1.Conclusions: Our results indicated that PBA might be a new Nrf2 activator that covalently binds to two critical domains of Keap1, and shows cardioprotective activities against ischemia-reperfusion injury.

Persulfide Signaling in Stress-Initiated Calmodulin Kinase Response

Significance: Calcium ion (Ca2+)/calmodulin (CaM)-dependent protein kinases (CaMKs) are activated by phosphorylation of a crucial threonine residue either by itself (CaMKII) or by upstream kinases, CaMK kinases (CaMKKs) (CaMKI and CaMKIV). CaMKs, present in most mammalian tissues, can phosphorylate many downstream targets, thereby regulating numerous cellular functions. Recent Advances: Aside from canonical post-translational modifications, cysteine-based redox switches in CaMKs affect their enzyme activities. In addition to reactive oxygen species (ROS) and reactive nitrogen species (RNS), reactive sulfur species (RSS) are also recognized as key signaling molecules, regulating protein function through polysulfidation, formation of polysulfides [-S-(S)n-H] on their reactive cysteine residues. To comprehend the biological significance of RSS signaling-related CaMK regulation, here we introduce a novel concept defining CaMKs as RSS targets in stress responses. The stress responses include an irreversible electrophile attack for CaMKI, inflammation for CaMKII, and endoplasmic reticulum stress for CaMKIV. Critical Issues: Development of various human diseases is associated with increased ROS, RNS, and RSS generation. Therefore, depending on specific pathophysiology, RSS could have very particular effects on CaMK functions. Future Directions: How multiple sources and mutual reactions of ROS, RNS, and RSS are coordinated is obscure. Elucidating the mechanisms through applications of enzymology, chemical biology, and mass spectrometry enables to uncover the complexities of redox regulation of CaMK cascades.

Irritant-evoked activation and calcium modulation of the TRPA1 receptor.

The TRPA1 ion channel is expressed by primary afferent nerve fibers, where it functions as a low threshold sensor for structurally diverse electrophilic irritants ranging from small volatile environmental toxicants to endogenous algogenic lipids1. TRPA1 is also a ‘receptor-operated’ channel whose activation downstream of metabotropic receptors elicits inflammatory pain or itch, making it an attractive target for novel analgesic therapies2. However, we lack mechanistic insight into how TRPA1 recognizes and responds to electrophiles or cytoplasmic second messengers. Here, we show that electrophiles act through a two-step process in which modification of a highly reactive cysteine (C621) promotes reorientation of a cytoplasmic loop to enhance nucleophilicity and modification of a nearby cysteine (C665), thereby stabilizing the loop in an activating configuration. These actions modulate two restrictions controlling ion permeation, including widening of the selectivity filter to enhance calcium permeability and opening of a canonical gate at the cytoplasmic end of the pore. We propose a model to explain functional coupling between electrophile action and these control points. We also characterize a calcium binding pocket that is remarkably conserved across TRP channel subtypes and accounts for all aspects of calcium-dependent TRPA1 regulation, including potentiation, desensitization, and activation by metabotropic receptors. These findings provide a structural framework for understanding how a broad-spectrum irritant receptor is controlled by endogenous and exogenous agents that elicit or exacerbate pain and itch.

Dysregulated hydrogen sulfide metabolism links aberrant neuronal stress responses and neurodegeneration in Huntington's disease

Hydrogen sulfide (H2S) is a gaseous signaling molecule that plays central roles in a myriad of physiological processes ranging from response to oxidative stress and transcriptional regulation. Hydrogen sulfide is synthesized endogenously from the semi‐essential amino acid cysteine via the reverse transsulfuration pathway. Thus disrupted cysteine metabolism may also lead to aberrant H2S signaling. One of the modes by which hydrogen sulfide signals is by a posttranslational modification designated as sulfhydration/persulfidation, which occurs on reactive cysteine residues on target proteins, where the reactive SH group is converted to an SSH group. Sulfhydration is a highly prevalent modification which modifies the structure and/or activity of target proteins. Research on H2S and persulfidation is still in its infancy. We show here that H2S is generated in response to stress stimuli such as Golgi stress and oxidative stress in neurons, which modulates sulfhydration levels to modulate signaling pathways. We further show that these cytoprotective stress responses are compromised in striatal cell culture model and the R6/2 and Q175 mouse models of Huntington’s disease (HD). We have shown previously that the biosynthetic enzyme for cysteine and H2S in neurons, cystathionine g‐lyase (CSE) is depleted in HD and mediates neurodegeneraton. HD is a neurodegenerative disease which is triggered by expansion of polyglutamine repeats in the protein huntingtin, which causes it to aggregate and elicit multi‐system toxicity. Restoring H2S balance in cells mitigates neuronal dysfunction in neurodegenerative diseases such as HD. A screen for regulators of the transsulfuration pathway has resulted in identification of compounds that modulate cysteine balance and disease progression in HD.Support or Funding InformationUnited States Public Health Service (USPHS) grants DA000266 and MH18501 to S.H.S.

Hydrogels With Tunable Mechanical Properties Based on Photocleavable Proteins.

Hydrogels with photo-responsive mechanical properties have found broad biomedical applications, including delivering bioactive molecules, cell culture, biosensing, and tissue engineering. Here, using a photocleavable protein, PhoCl, as the crosslinker we engineer two types of poly(ethylene glycol) hydrogels whose mechanical stability can be weakened or strengthened, respectively, upon visible light illumination. In the photo weakening hydrogels, photocleavage leads to rupture of the protein crosslinkers, and decrease of the mechanical properties of the hydrogels. In contrast, in the photo strengthening hydrogels, by properly choosing the crosslinking positions, photocleavage does not rupture the crosslinking sites but exposes additional cryptical reactive cysteine residues. When reacting with extra maleimide groups in the hydrogel network, the mechanical properties of the hydrogels can be enhanced upon light illumination. Our study indicates that photocleavable proteins could provide more designing possibilities than the small-molecule counterparts. A proof-of-principle demonstration of spatially controlling the mechanical properties of hydrogels was also provided.

Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum.

The endoplasmic reticulum (ER) is the initial site of biogenesis of secretory pathway proteins, including proteins localized to the ER, Golgi, lysosomes, intracellular vesicles, plasma membrane, and extracellular compartments. Proteins within the secretory pathway contain a high abundance of disulfide bonds to protect against the oxidative extracellular environment. These disulfide bonds are typically formed within the ER by a variety of oxidoreductases, including members of the protein disulfide isomerase (PDI) family. Here, we establish chemoproteomic platforms to identify oxidized and reduced cysteine residues within the ER. Subcellular fractionation methods were utilized to enrich for the ER and significantly enhance the coverage of ER-localized cysteine residues. Reactive-cysteine profiling ranked ∼900 secretory pathway cysteines by reactivity with an iodoacetamide-alkyne probe, revealing functional cysteines annotated to participate in disulfide bonds, or S-palmitoylation sites within proteins. Through application of a variation of the OxICAT protocol for quantifying cysteine oxidation, the percentages of oxidation for each of ∼700 ER-localized cysteines were calculated. Lastly, perturbation of ER function, through chemical induction of ER stress, was used to investigate the effect of initiation of the unfolded protein response (UPR) on ER-localized cysteine oxidation. Together, these studies establish a platform for identifying reactive and functional cysteine residues on proteins within the secretory pathway as well as for interrogating the effects of diverse cellular stresses on ER-localized cysteine oxidation.

An anticancer gold(III)-activated porphyrin scaffold that covalently modifies protein cysteine thiols

Cysteine thiols of many cancer-associated proteins are attractive targets of anticancer agents. Herein, we unequivocally demonstrate a distinct thiol-targeting property of gold(III) mesoporphyrin IX dimethyl ester (AuMesoIX) and its anticancer activities. While the binding of cysteine thiols with metal complexes usually occurs via M-S bond formation, AuMesoIX is unique in that the meso-carbon atom of the porphyrin ring is activated by the gold(III) ion to undergo nucleophilic aromatic substitution with thiols. AuMesoIX was shown to modify reactive cysteine residues and inhibit the activities of anticancer protein targets including thioredoxin, peroxiredoxin, and deubiquitinases. Treatment of cancer cells with AuMesoIX resulted in the formation of gold-bound sulfur-rich protein aggregates, oxidative stress-mediated cytotoxicity, and accumulation of ubiquitinated proteins. Importantly, AuMesoIX exhibited effective antitumor activity in mice. Our study has uncovered a gold(III)-induced ligand scaffold reactivity for thiol targeting that can be exploited for anticancer applications.

PRL2 serves as a negative regulator in cell adaptation to oxidative stress

High levels of ROS cause oxidative stress, which plays a critical role in cell death. As a ROS effector protein, PRL2 senses ROS and controls phagocyte bactericidal activity during infection. Here we report PRL2 regulates oxidative stress induced cell death. PRL2 senses oxidative stress via highly reactive cysteine residues at 46 and 101. The oxidation of PRL2 causes protein degradation and supports pro-survival PDK1/AKT signal which in turn to protect cells against oxidative stress. As a result, PRL2 levels have a high correlation with oxidative stress induced cell death. In vivo experiments showed PRL2 deficient cells survive better in inflammatory oxidative environment and resist to ionizing radiation. Our finding suggests PRL2 serves as a negative regulator in cell adaptation to oxidative stress. Therefore, PRL2 could be targeted to modulate cell viability in inflammation or irradiation associated therapy.

Cysteine-targeted Irreversible Inhibitors of Tyrosine Kinases and Key Interactions.

Tyrosine kinases are a subgroup of a large class of protein kinases that transfer phosphate groups from ATP to various amino acid residues. By phosphorylating the tyrosine residues, the tyrosine kinases are responsible for the activation of various proteins through signal transduction cascades, which serves as a ubiquitous mechanism of cell signaling. The frequent success of many tyrosine kinase inhibitors (TKIs) in clinical success and diseasecausing mutations in protein kinases suggests that a large number of kinases may represent therapeutically relevant targets. To date, most of the clinical and preclinical TKIs are ATPcompetitive non-covalent inhibitors, which achieve their selectivity by recognizing the unique features of specific protein kinases. Of growing interest now in the scientific community is the development of irreversible inhibitors that form covalent bonds with cysteines or other nucleophilic residues in the ATP binding pocket. Irreversible TKIs have many potential advantages including prolonged pharmacodynamics, reasonable compound design suitability, high potency, and the ability to validate pharmacological specificity by mutations in reactive cysteine residues. Here, we review recent efforts to develop cysteine-targeting irreversible TKIs and to discuss their patterns of configuration that identify adenosine triphosphate binding pockets and their biological activities.

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 forchemoselective persulfide bioconjugation usingdimedone-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 longevityand improved capacity to cope with stress stimuli.