Zinc-chelating BET bromodomain inhibitors equally target islet endocrine cell types.

Inhibition of the bromodomain and extraterminal domain (BET) protein family is a potential strategy to prevent and treat diabetes; however, the clinical use of BET bromodomain inhibitors (BETis) is associated with adverse effects. Here, we explore a strategy for targeting BETis to ß cells by exploiting the high-zinc (Zn2+) concentration in ß cells relative to other cell types. We report the synthesis of a novel, Zn2+-chelating derivative of the pan-BETi (+)-JQ1, (+)-JQ1-DPA, in which (+)-JQ1 was conjugated to dipicolyl amine (DPA). As controls, we synthesized (+)-JQ1-DBA, a non-Zn2+-chelating derivative, and (-)-JQ1-DPA, an inactive enantiomer that chelates Zn2+. Molecular modeling and biophysical assays showed that (+)-JQ1-DPA and (+)-JQ1-DBA retain potent binding to BET bromodomains in vitro. Cellular assays demonstrated (+)-JQ1-DPA attenuated NF-ĸB target gene expression in ß cells stimulated with the proinflammatory cytokine interleukin 1ß. To assess ß-cell selectivity, we isolated islets from a mouse model that expresses green fluorescent protein in insulin-positive ß cells and mTomato in insulin-negative cells (non-ß cells). Surprisingly, Zn2+ chelation did not confer ß-cell selectivity as (+)-JQ1-DPA was equally effective in both ß and α cells; however, (+)-JQ1-DPA was less effective in macrophages, a nonendocrine islet cell type. Intriguingly, the non-Zn2+-chelating derivative (+)-JQ1-DBA displayed the opposite selectivity, with greater effect in macrophages compared with (+)-JQ1-DPA, suggesting potential as a macrophage-targeting molecule. These findings suggest that Zn2+-chelating small molecules confer endocrine cell selectivity rather than ß-cell selectivity in pancreatic islets and provide valuable insights and techniques to assess Zn2+ chelation as an approach to selectively target small molecules to pancreatic ß cells.NEW & NOTEWORTHY Inhibition of BET bromodomains is a novel potential strategy to prevent and treat diabetes mellitus. However, BET inhibitors have negative side effects. We synthesized a BET inhibitor expected to exploit the high zinc concentration in ß cells to accumulate in ß cells. We show our inhibitor targeted pancreatic endocrine cells; however, it was less effective in immune cells. A control inhibitor showed the opposite effect. These findings help us understand how to target specific cells in diabetes treatment.

Identification of Protein Targets of S-Nitroso-Coenzyme A-Mediated S-Nitrosation Using Chemoproteomics.

S-Nitrosation is a cysteine post-translational modification fundamental to cellular signaling. This modification regulates protein function in numerous biological processes in the nervous, cardiovascular, and immune systems. Small molecule or protein nitrosothiols act as mediators of NO signaling by transferring the NO group (formally NO+) to a free thiol on a target protein through a transnitrosation reaction. The protein targets of specific transnitrosating agents and the extent and functional effects of S-nitrosation on these target proteins have been poorly characterized. S-nitroso-coenzyme A (CoA-SNO) was recently identified as a mediator of endogenous S-nitrosation. Here, we identified direct protein targets of CoA-SNO-mediated transnitrosation using a competitive chemical-proteomic approach that quantified the extent of modification on 789 cysteine residues in response to CoA-SNO. A subset of cysteines displayed high susceptibility to modification by CoA-SNO, including previously uncharacterized sites of S-nitrosation. We further validated and functionally characterized the functional effects of S-nitrosation on the protein targets phosphofructokinase (platelet type), ATP citrate synthase, and ornithine aminotransferase.

N-terminal BET bromodomain inhibitors disrupt a BRD4-p65 interaction and reduce inducible nitric oxide synthase transcription in pancreatic ß-cells.

Chronic inflammation of pancreatic islets is a key driver of ß-cell damage that can lead to autoreactivity and the eventual onset of autoimmune diabetes (T1D). In the islet, elevated levels of proinflammatory cytokines induce the transcription of the inducible nitric oxide synthase (iNOS) gene, NOS2, ultimately resulting in increased nitric oxide (NO). Excessive or prolonged exposure to NO causes ß-cell dysfunction and failure associated with defects in mitochondrial respiration. Recent studies showed that inhibition of the bromodomain and extraterminal domain (BET) family of proteins, a druggable class of epigenetic reader proteins, prevents the onset and progression of T1D in the non-obese diabetic mouse model. We hypothesized that BET proteins co-activate transcription of cytokine-induced inflammatory gene targets in ß-cells and that selective, chemotherapeutic inhibition of BET bromodomains could reduce such transcription. Here, we investigated the ability of BET bromodomain small molecule inhibitors to reduce the ß-cell response to the proinflammatory cytokine interleukin 1 beta (IL-1ß). BET bromodomain inhibition attenuated IL-1ß-induced transcription of the inflammatory mediator NOS2 and consequent iNOS protein and NO production. Reduced NOS2 transcription is consistent with inhibition of NF-κB facilitated by disrupting the interaction of a single BET family member, BRD4, with the NF-κB subunit, p65. Using recently reported selective inhibitors of the first and second BET bromodomains, inhibition of only the first bromodomain was necessary to reduce the interaction of BRD4 with p65 in ß-cells. Moreover, inhibition of the first bromodomain was sufficient to mitigate IL-1ß-driven decreases in mitochondrial oxygen consumption rates and ß-cell viability. By identifying a role for the interaction between BRD4 and p65 in controlling the response of ß-cells to proinflammatory cytokines, we provide mechanistic information on how BET bromodomain inhibition can decrease inflammation. These studies also support the potential therapeutic application of more selective BET bromodomain inhibitors in attenuating ß-cell inflammation.

Sirtuin Oxidative Post-translational Modifications.

Increased sirtuin deacylase activity is correlated with increased lifespan and healthspan in eukaryotes. Conversely, decreased sirtuin deacylase activity is correlated with increased susceptibility to aging-related diseases. However, the mechanisms leading to decreased sirtuin activity during aging are poorly understood. Recent work has shown that oxidative post-translational modification by reactive oxygen (ROS) or nitrogen (RNS) species results in inhibition of sirtuin deacylase activity through cysteine nitrosation, glutathionylation, sulfenylation, and sulfhydration as well as tyrosine nitration. The prevalence of ROS/RNS (e.g., nitric oxide, S-nitrosoglutathione, hydrogen peroxide, oxidized glutathione, and peroxynitrite) is increased during inflammation and as a result of electron transport chain dysfunction. With age, cellular production of ROS/RNS increases; thus, cellular oxidants may serve as a causal link between loss of sirtuin activity and aging-related disease development. Therefore, the prevention of inhibitory oxidative modification may represent a novel means to increase sirtuin activity during aging. In this review, we explore the role of cellular oxidants in inhibiting individual sirtuin human isoform deacylase activity and clarify the relevance of ROS/RNS as regulatory molecules of sirtuin deacylase activity in the context of health and disease.

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.

Human sirtuins are differentially sensitive to inhibition by nitrosating agents and other cysteine oxidants.

Sirtuins (e.g. human Sirt1-7) catalyze the removal of acyl groups from lysine residues in proteins in an NAD+-dependent manner, and loss of sirtuin deacylase activity correlates with the development of aging-related diseases. Although multiple reports suggest that sirtuin activity is regulated by oxidative post-translational modifications of cysteines during inflammation and aging, no systematic comparative study of potential direct sirtuin cysteine oxidative modifications has been performed. Here, using IC50 and kinact/KI analyses, we quantified the ability of nitrosothiols (S-nitrosoglutathione and S-nitroso-N-acetyl-d,l-penicillamine), nitric oxide, oxidized GSH, and hydrogen peroxide to post-translationally modify and inhibit the deacylase activity of Sirt1, Sirt2, Sirt3, Sirt5, and Sirt6. The inhibition was correlated with cysteine modification and assessed with chemical-probe and blot-based assays for cysteine S-nitrosation, sulfenylation, and glutathionylation. We show that the primarily nuclear sirtuins Sirt1 and Sirt6, as well as the primarily cytosolic sirtuin Sirt2, are modified and inhibited by cysteine S-nitrosation in response to exposure to both free nitric oxide and nitrosothiols (kinact/KI ≥ 5 m-1 s-1), which is the first report of Sirt2 and Sirt6 inhibition by S-nitrosation. Surprisingly, the mitochondrial sirtuins Sirt3 and Sirt5 were resistant to inhibition by cysteine oxidants. Collectively, these results suggest that nitric oxide-derived oxidants may causatively link nuclear and cytosolic sirtuin inhibition to aging-related inflammatory disease development.

Covalent-Fragment Screening of BRD4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites.

BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to nonhomologous cysteine residues within the C-terminal BRD4 bromodomain (BRD4-BD2), we performed a midthroughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify BRD4. Subsequent mass spectrometry, NMR, and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to BRD4 among human bromodomains. This site is orthogonal to the BRD4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays nor an acetylated histone peptide in AlphaScreen assays. Finally, we tethered our top-performing covalent fragment to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace BRD4 from chromatin.

Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically relevant metabolic enzymes.

Cysteine S-nitrosation is a reversible post-translational modification mediated by nitric oxide (•NO)-derived agents. S-Nitrosation participates in cellular signaling and is associated with several diseases such as cancer, cardiovascular diseases, and neuronal disorders. Despite the physiological importance of this nonclassical •NO-signaling pathway, little is understood about how much S-nitrosation affects protein function. Moreover, identifying physiologically relevant targets of S-nitrosation is difficult because of the dynamics of transnitrosation and a limited understanding of the physiological mechanisms leading to selective protein S-nitrosation. To identify proteins whose activities are modulated by S-nitrosation, we performed a metabolomics study comparing WT and endothelial nitric-oxide synthase knockout mice. We integrated our results with those of a previous proteomics study that identified physiologically relevant S-nitrosated cysteines, and we found that the activity of at least 21 metabolic enzymes might be regulated by S-nitrosation. We cloned, expressed, and purified four of these enzymes and observed that S-nitrosation inhibits the metabolic enzymes 6-phosphogluconate dehydrogenase, Δ1-pyrroline-5-carboxylate dehydrogenase, catechol-O-methyltransferase, and d-3-phosphoglycerate dehydrogenase. Furthermore, using site-directed mutagenesis, we identified the predominant cysteine residue influencing the observed activity changes in each enzyme. In summary, using an integrated metabolomics approach, we have identified several physiologically relevant S-nitrosation targets, including metabolic enzymes, which are inhibited by this modification, and we have found the cysteines modified by S-nitrosation in each enzyme.

Nitrosothiol formation and S-nitrosation signaling through nitric oxide synthases.

Nitric oxide (NO) is a gaseous signaling molecule impacting many biological pathways. NO is produced in mammals by three nitric oxide synthase (NOS) isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). nNOS and eNOS produce low concentrations of NO for paracrine signaling; NO produced and released from one cell diffuses to a neighboring cell where it binds and activates soluble guanylyl cyclase (sGC). iNOS produces high concentrations of NO using NO toxicity to amplify the innate immune response. Recent work has also defined protein cysteine S-nitrosation as a pathway of sGC-independent NO signaling. Though many studies have shown that S-nitrosation regulates the activity of NOS isoforms and other proteins in vivo, many issues need to be resolved to establish S-nitrosation as a viable signaling mechanism. Several chemical mechanisms result in S-nitrosation including transition metal-catalyzed pathways, NO oxidation followed by thiolate reaction, and thiyl radical recombination with NO. Once formed, nitrosothiols can be transferred between cellular cysteine residues via transnitrosation reactions. However, it is largely unclear how these chemical processes result in selective S-nitrosation of specific cellular cysteine residues. S-nitrosation site selectivity may be imparted via direct interactions or colocalization with NOS isoforms that focus chemical or transnitrosation mechanisms of nitrosothiol formation or transfer. Here, we discuss chemical mechanisms of nitrosothiol formation, S-nitrosation of NOS isoforms, and potential S-nitrosation signaling cascades resulting from NOS S-nitrosation.

Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation.

The sirtuin family of proteins catalyze the NAD+-dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1-7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD+ and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn2+ release from the conserved sirtuin Zn2+-tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD+ and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn2+, consistent with S-nitrosation of the Zn2+-tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment.

Chemoproteomic Strategy to Quantitatively Monitor Transnitrosation Uncovers Functionally Relevant S-Nitrosation Sites on Cathepsin D and HADH2.

S-Nitrosoglutathione (GSNO) is an endogenous transnitrosation donor involved in S-nitrosation of a variety of cellular proteins, thereby regulating diverse protein functions. Quantitative proteomic methods are necessary to establish which cysteine residues are most sensitive to GSNO-mediated transnitrosation. Here, a competitive cysteine-reactivity profiling strategy was implemented to quantitatively measure the sensitivity of >600 cysteine residues to transnitrosation by GSNO. This platform identified a subset of cysteine residues with a high propensity for GSNO-mediated transnitrosation. Functional characterization of previously unannotated S-nitrosation sites revealed that S-nitrosation of a cysteine residue distal to the 3-hydroxyacyl-CoA dehydrogenase type 2 (HADH2) active site impaired catalytic activity. Similarly, S-nitrosation of a non-catalytic cysteine residue in the lysosomal aspartyl protease cathepsin D (CTSD) inhibited proteolytic activation. Together, these studies revealed two previously uncharacterized cysteine residues that regulate protein function, and established a chemical-proteomic platform with capabilities to determine substrate specificity of other cellular transnitrosation agents.

Truncating mutation in the nitric oxide synthase 1 gene is associated with infantile achalasia.

Nitric oxide is thought to have a role in the pathogenesis of achalasia. We performed a genetic analysis of 2 siblings with infant-onset achalasia. Exome analysis revealed that they were homozygous for a premature stop codon in the gene encoding nitric oxide synthase 1. Kinetic analyses and molecular modeling showed that the truncated protein product has defects in folding, nitric oxide production, and binding of cofactors. Heller myotomy had no effect in these patients, but sildenafil therapy increased their ability to drink. The finding recapitulates the previously reported phenotype of nitric oxide synthase 1-deficient mice, which have achalasia. Nitric oxide signaling appears to be involved in the pathogenesis of achalasia in humans.

Electrostatic ratchet in the protective antigen channel promotes anthrax toxin translocation.

Central to the power-stroke and brownian-ratchet mechanisms of protein translocation is the process through which nonequilibrium fluctuations are rectified or ratcheted by the molecular motor to transport substrate proteins along a specific axis. We investigated the ratchet mechanism using anthrax toxin as a model. Anthrax toxin is a tripartite toxin comprised of the protective antigen (PA) component, a homooligomeric transmembrane translocase, which translocates two other enzyme components, lethal factor (LF) and edema factor (EF), into the cytosol of the host cell under the proton motive force (PMF). The PA-binding domains of LF and EF (LF(N) and EF(N)) possess identical folds and similar solution stabilities; however, EF(N) translocates ∼10-200-fold slower than LF(N), depending on the electrical potential (Δψ) and chemical potential (ΔpH) compositions of the PMF. From an analysis of LF(N)/EF(N) chimera proteins, we identified two 10-residue cassettes comprised of charged sequence that were responsible for the impaired translocation kinetics of EF(N). These cassettes have nonspecific electrostatic requirements: one surprisingly prefers acidic residues when driven by either a Δψ or a ΔpH; the second requires basic residues only when driven by a Δψ. Through modeling and experiment, we identified a charged surface in the PA channel responsible for charge selectivity. The charged surface latches the substrate and promotes PMF-driven transport. We propose an electrostatic ratchet in the channel, comprised of opposing rings of charged residues, enforces directionality by interacting with charged cassettes in the substrate, thereby generating forces sufficient to drive unfolding.

hERG gating microdomains defined by S6 mutagenesis and molecular modeling.

Human ether-à-go-go-related gene (hERG) channels mediate cardiac repolarization and bind drugs that can cause acquired long QT syndrome and life-threatening arrhythmias. Drugs bind in the vestibule formed by the S6 transmembrane domain, which also contains the activation gate that traps drugs in the vestibule and contributes to their efficacy of block. Although drug-binding residues have been identified, we know little about the roles of specific S6 residues in gating. We introduced cysteine mutations into the hERG channel S6 domain and measured mutational effects on the steady-state distribution and kinetics of transitions between the closed and open states. Energy-minimized molecular models based on the crystal structures of rKv1.2 (open state) and MlotiK1 and KcsA (closed state) provided structural contexts for evaluating mutant residues. The majority of mutations slowed deactivation, shifted conductance voltage curves to more negative potentials, or conferred a constitutive conductance over voltages that normally cause the channel to close. At the most intracellular extreme of the S6 region, Q664, Y667, and S668 were especially sensitive and together formed a ringed domain that occludes the pore in the closed state model. In contrast, mutation of S660, more than a full helical turn away and corresponding by alignment to a critical Shaker gate residue (V478), had little effect on gating. Multiple substitutions of chemically distinct amino acids at the adjacent V659 suggested that, upon closing, the native V659 side chain moves into a hydrophobic pocket but likely does not form the occluding gate itself. Overall, the study indicated that S6 mutagenesis disrupts the energetics primarily of channel closing and identified several residues critical for this process in the native channel.