Covalent Modification Of Proteins Research Articles

Inhibition of Autophagy by a Small Molecule through Covalent Modification of the LC3 Protein

AbstractThe autophagic ubiquitin‐like protein LC3 functions through interactions with LC3‐interaction regions (LIRs) of other autophagy proteins, including autophagy receptors, which stands out as a promising protein–protein interaction (PPI) target for the intervention of autophagy. Post‐translational modifications like acetylation of Lys49 on the LIR‐interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering with the interface. Through screening covalent compounds, we discovered a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49. Activity‐based protein profiling (ABPP) based evaluations reveal that a derivative molecule DC‐LC3in‐D5 exhibits a potent covalent reactivity and selectivity to LC3A/B in HeLa cells. DC‐LC3in‐D5 compromises LC3B lipidation in vitro and in HeLa cells, leading to deficiency in the formation of autophagic structures and autophagic substrate degradation. DC‐LC3in‐D5 could serve as a powerful tool for autophagy research as well as for therapeutic interventions.

Inhibition of Autophagy by a Small Molecule through Covalent Modification of the LC3 Protein.

The autophagic ubiquitin-like protein LC3 functions through interactions with LC3-interaction regions (LIRs) of other autophagy proteins, including autophagy receptors, which stands out as a promising protein-protein interaction (PPI) target for the intervention of autophagy. Post-translational modifications like acetylation of Lys49 on the LIR-interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering with the interface. Through screening covalent compounds, we discovered a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49. Activity-based protein profiling (ABPP) based evaluations reveal that a derivative molecule DC-LC3in-D5 exhibits a potent covalent reactivity and selectivity to LC3A/B in HeLa cells. DC-LC3in-D5 compromises LC3B lipidation in vitro and in HeLa cells, leading to deficiency in the formation of autophagic structures and autophagic substrate degradation. DC-LC3in-D5 could serve as a powerful tool for autophagy research as well as for therapeutic interventions.

Extensive Anti-CoA Immunostaining in Alzheimer's Disease and Covalent Modification of Tau by a Key Cellular Metabolite Coenzyme A.

Alzheimer’s disease (AD) is a neurodegenerative disorder, accounting for at least two-thirds of dementia cases. A combination of genetic, epigenetic and environmental triggers is widely accepted to be responsible for the onset and development of AD. Accumulating evidence shows that oxidative stress and dysregulation of energy metabolism play an important role in AD pathogenesis, leading to neuronal dysfunction and death. Redox-induced protein modifications have been reported in the brain of AD patients, indicating excessive oxidative damage. Coenzyme A (CoA) is essential for diverse metabolic pathways, regulation of gene expression and biosynthesis of neurotransmitters. Dysregulation of CoA biosynthesis in animal models and inborn mutations in human genes involved in the CoA biosynthetic pathway have been associated with neurodegeneration. Recent studies have uncovered the antioxidant function of CoA, involving covalent protein modification by this cofactor (CoAlation) in cellular response to oxidative or metabolic stress. Protein CoAlation has been shown to both modulate the activity of modified proteins and protect cysteine residues from irreversible overoxidation. In this study, immunohistochemistry analysis with highly specific anti-CoA monoclonal antibody was used to reveal protein CoAlation across numerous neurodegenerative diseases, which appeared particularly frequent in AD. Furthermore, protein CoAlation consistently co-localized with tau-positive neurofibrillary tangles, underpinning one of the key pathological hallmarks of AD. Double immunihistochemical staining with tau and CoA antibodies in AD brain tissue revealed co-localization of the two immunoreactive signals. Further, recombinant 2N3R and 2N4R tau isoforms were found to be CoAlated in vitro and the site of CoAlation mapped by mass spectrometry to conserved cysteine 322, located in the microtubule binding region. We also report the reversible H2O2-induced dimerization of recombinant 2N3R, which is inhibited by CoAlation. Moreover, CoAlation of transiently expressed 2N4R tau was observed in diamide-treated HEK293/Pank1β cells. Taken together, this study demonstrates for the first time extensive anti-CoA immunoreactivity in AD brain samples, which occurs in structures resembling neurofibrillary tangles and neuropil threads. Covalent modification of recombinant tau at cysteine 322 suggests that CoAlation may play an important role in protecting redox-sensitive tau cysteine from irreversible overoxidation and may modulate its acetyltransferase activity and functional interactions.

Glutathione Metabolism in Plants under Stress: Beyond Reactive Oxygen Species Detoxification.

Glutathione is an essential metabolite for plant life best known for its role in the control of reactive oxygen species (ROS). Glutathione is also involved in the detoxification of methylglyoxal (MG) which, much like ROS, is produced at low levels by aerobic metabolism under normal conditions. While several physiological processes depend on ROS and MG, a variety of stresses can dramatically increase their concentration leading to potentially deleterious effects. In this review, we examine the structure and the stress regulation of the pathways involved in glutathione synthesis and degradation. We provide a synthesis of the current knowledge on the glutathione-dependent glyoxalase pathway responsible for MG detoxification. We present recent developments on the organization of the glyoxalase pathway in which alternative splicing generate a number of isoforms targeted to various subcellular compartments. Stress regulation of enzymes involved in MG detoxification occurs at multiple levels. A growing number of studies show that oxidative stress promotes the covalent modification of proteins by glutathione. This post-translational modification is called S-glutathionylation. It affects the function of several target proteins and is relevant to stress adaptation. We address this regulatory function in an analysis of the enzymes and pathways targeted by S-glutathionylation.

SUMO and SUMOylation Pathway at the Forefront of Host Immune Response.

Pathogens pose a continuous challenge for the survival of the host species. In response to the pathogens, the host immune system mounts orchestrated defense responses initiating various mechanisms both at the cellular and molecular levels, including multiple post-translational modifications (PTMs) leading to the initiation of signaling pathways. The network of such pathways results in the recruitment of various innate immune components and cells at the site of infection and activation of the adaptive immune cells, which work in synergy to combat the pathogens. Ubiquitination is one of the most commonly used PTMs. Host cells utilize ubiquitination for both temporal and spatial regulation of immune response pathways. Over the last decade, ubiquitin family proteins, particularly small ubiquitin-related modifiers (SUMO), have been widely implicated in host immune response. SUMOs are ubiquitin-like (Ubl) proteins transiently conjugated to a wide variety of proteins through SUMOylation. SUMOs primarily exert their effect on target proteins by covalently modifying them. However, SUMO also engages in a non-covalent interaction with the SUMO-interacting motif (SIM) in target proteins. Unlike ubiquitination, SUMOylation alters localization, interactions, functions, or stability of target proteins. This review provides an overview of the interplay of SUMOylation and immune signaling and development pathways in general. Additionally, we discuss in detail the regulation exerted by covalent SUMO modifications of target proteins, and SIM mediated non-covalent interactions with several effector proteins. In addition, we provide a comprehensive review of the literature on the importance of the SUMO pathway in the development and maintenance of a robust immune system network of the host. We also summarize how pathogens modulate the host SUMO cycle to sustain infectability. Studies dealing mainly with SUMO pathway proteins in the immune system are still in infancy. We anticipate that the field will see a thorough and more directed analysis of the SUMO pathway in regulating different cells and pathways of the immune system. Our current understanding of the importance of the SUMO pathway in the immune system necessitates an urgent need to synthesize specific inhibitors, bioactive regulatory molecules, as novel therapeutic targets.

Oxidative-Stress-Induced Cellular Toxicity and Glycoxidation of Biomolecules by Cosmetic Products under Sunlight Exposure

Cosmetics, commonly known as ‘makeup’ are products that can enhance the appearance of the human body. Cosmetic products include hair dyes, shampoos, skincare, sunscreens, kajal, and other makeup products. Cosmetics are generally applied throughout the face and over the neck region. Sunlight has different wavelengths of light, which include UV-A, UV-B, UV-C, and other radiations. Most cosmetic products have absorption maxima (λmax) in the range of visible light and UV-R. The effect of light-induced photosensitization of cosmetic products, which results in the production of free radicals through type-I and type-II photosensitization mechanisms. Free-radicals-mediated DNA damage and oxidative stress are common consequences of cosmetic phototoxicity. Cosmetic phototoxicity may include percutaneous absorption, skin irritation, eye irritation, photosensitization, mutagenicity, and genotoxicity. Oxidative stress induces membrane lipid peroxidation, glycoxidation, and protein covalent modifications, resulting in their dysfunction. Natural antioxidants inhibit oxidative-stress-induced cosmetic toxicity. Sunlight-induced photodegradation and accumulation of cosmetic photoproducts are also a matter of serious concern. India has tropical weather conditions throughout the year and generally, a majority of human activities such as commerce, agriculture, sports, etc. are performed under bright sunlight conditions. Thus, more focused and dedicated research is warranted to explore the effects of cosmetics on oxidative stress, glycoxidation of biomolecules, and photoproducts accumulation for its total human safety.

Characterization of Histone H3 Lysine 4 and 36 Tri-methylation in Brassica rapa L.

Covalent modifications of histone proteins act as epigenetic regulators of gene expression. We report the distribution of two active histone marks (H3K4me3 and H3K36me3) in 14-day leaves in two lines of Brassica rapa L. by chromatin immunoprecipitation sequencing. Both lines were enriched with H3K4me3 and H3K36me3 marks at the transcription start site, and the transcription level of a gene was associated with the level of H3K4me3 and H3K36me3. H3K4me3- and H3K36me3-marked genes showed low tissue-specific gene expression, and genes with both H3K4me3 and H3K36me3 had a high level of expression and were constitutively expressed. Bivalent active and repressive histone modifications such as H3K4me3 and H3K27me3 marks or antagonistic coexistence of H3K36me3 and H3K27me3 marks were observed in some genes. Expression may be susceptible to changes by abiotic and biotic stresses in genes having both H3K4me3 and H3K27me3 marks. We showed that the presence of H3K36me3 marks was associated with different gene expression levels or tissue specificity between paralogous paired genes, suggesting that H3K36me3 might be involved in subfunctionalization of the subgenomes.

EGFR in Cancer: Signaling Mechanisms, Drugs, and Acquired Resistance.

Simple SummaryGrowth factors are hormone-like molecules able to promote division and migration of normal cells, but cancer captured the underlying mechanisms to unleash tumor growth and metastasis. Here we review the epidermal growth factor (EGF), which controls epithelial cells, the precursors of all carcinomas, and the cognate cell surface receptor, called EGFR. In addition to over-production of EGF and its family members in tumors, EGFR is similarly over-produced, and mutant hyper-active forms of EGFR are uniquely found in some brain, lung, and other cancers. After describing the biochemical mechanisms underlying the cancer-promoting actions of EGFR, we review some of the latest research discoveries and list all anti-cancer drugs specifically designed to block the EGFR’s biochemical pathway. We conclude by explaining why some patients with lung or colorectal cancer do not respond to the anti-EGFR therapies and why still other patients, who initially respond, become tolerant to the drugs.The epidermal growth factor receptor (EGFR) has served as the founding member of the large family of growth factor receptors harboring intrinsic tyrosine kinase function. High abundance of EGFR and large internal deletions are frequently observed in brain tumors, whereas point mutations and small insertions within the kinase domain are common in lung cancer. For these reasons EGFR and its preferred heterodimer partner, HER2/ERBB2, became popular targets of anti-cancer therapies. Nevertheless, EGFR research keeps revealing unexpected observations, which are reviewed herein. Once activated by a ligand, EGFR initiates a time-dependent series of molecular switches comprising downregulation of a large cohort of microRNAs, up-regulation of newly synthesized mRNAs, and covalent protein modifications, collectively controlling phenotype-determining genes. In addition to microRNAs, long non-coding RNAs and circular RNAs play critical roles in EGFR signaling. Along with driver mutations, EGFR drives metastasis in many ways. Paracrine loops comprising tumor and stromal cells enable EGFR to fuel invasion across tissue barriers, survival of clusters of circulating tumor cells, as well as colonization of distant organs. We conclude by listing all clinically approved anti-cancer drugs targeting either EGFR or HER2. Because emergence of drug resistance is nearly inevitable, we discuss the major evasion mechanisms.

PARP Power: A Structural Perspective on PARP1, PARP2, and PARP3 in DNA Damage Repair and Nucleosome Remodelling.

Poly (ADP-ribose) polymerases (PARP) 1-3 are well-known multi-domain enzymes, catalysing the covalent modification of proteins, DNA, and themselves. They attach mono- or poly-ADP-ribose to targets using NAD+ as a substrate. Poly-ADP-ribosylation (PARylation) is central to the important functions of PARP enzymes in the DNA damage response and nucleosome remodelling. Activation of PARP happens through DNA binding via zinc fingers and/or the WGR domain. Modulation of their activity using PARP inhibitors occupying the NAD+ binding site has proven successful in cancer therapies. For decades, studies set out to elucidate their full-length molecular structure and activation mechanism. In the last five years, significant advances have progressed the structural and functional understanding of PARP1-3, such as understanding allosteric activation via inter-domain contacts, how PARP senses damaged DNA in the crowded nucleus, and the complementary role of histone PARylation factor 1 in modulating the active site of PARP. Here, we review these advances together with the versatility of PARP domains involved in DNA binding, the targets and shape of PARylation and the role of PARPs in nucleosome remodelling.

USP15: a review of its implication in immune and inflammatory processes and tumor progression.

The covalent post-translational modification of proteins by ubiquitination not only influences protein stability and half-life, but also several aspects of protein function including enzymatic activity, sub-cellular localization, and interactions with binding partners. Protein ubiquitination status is determined by the action of large families of ubiquitin ligases and deubiquitinases, whose combined activities regulate many physiological and cellular pathways. The Ubiquitin Specific Protease (USP) family is one of 8 subfamilies of deubiquitinating enzymes composed of more than 50 members. Recent studies have shown that USP15 plays a critical role in regulating many aspects of immune and inflammatory function of leukocytes in response to a broad range of infectious and autoimmune insults and following tissue damage. USP15 regulated pathways reviewed herein include TLR signaling, RIG-I signaling, NF-kB, and IRF3/IRF7-dependent transcription for production of pro-inflammatory cytokines and type I interferons. In addition, USP15 has been found to regulate pathways implicated in tumor onset and progression such as p53, and TGF-β signaling, but also influences the leukocytes-determined immune and inflammatory microenvironment of tumors to affect progression and outcome. Hereby reviewed are recent studies of USP15 in model cell lines in vitro, and in mutant mice in vivo with reference to available human clinical datasets.

Influence of Dopamine on Fluorescent Advanced Glycation End Products Formation Using Drosophila melanogaster.

Non-enzymatic glycation and covalent modification of proteins leads to Advanced Glycation End products (AGEs). AGEs are biomarkers of aging and neurodegenerative disease, and can be induced by impaired neuronal signaling. The objective of this study was to investigate if manipulation of dopamine (DA) in vitro using the model protein, bovine serum albumin (BSA), and in vivo using the model organism Drosophila melanogaster, influences fluorescent AGEs (fAGEs) formation as an indicator of dopamine-induced oxidation events. DA inhibited fAGEs-BSA synthesis in vitro, suggesting an anti-oxidative effect, which was not observed when flies were fed DA. Feeding flies cocaine and methamphetamine led to increased fAGEs formation. Mutants lacking the dopaminergic transporter or the D1-type showed further elevation of fAGEs accumulation, indicating that the long-term perturbation in DA function leads to higher production of fAGEs. To confirm that DA has oxidative properties in vivo, we fed flies antioxidant quercetin (QUE) together with methamphetamine. QUE significantly decreased methamphetamine-induced fAGEs formation suggesting that the perturbation of DA function in vivo leads to increased oxidation. These findings present arguments for the use of fAGEs as a biomarker of DA-associated neurodegenerative changes and for assessment of antioxidant interventions such as QUE treatment.

4-Hydroxynonenal is An Oxidative Degradation Product of Polysorbate 80.

Polysorbates (PS) are used in biopharmaceuticals to stabilize therapeutic proteins. Oxidative degradation of (poly)unsaturated fatty acids (PUFAs) contained in PS was shown to lead to α,β-unsaturated carbonyls. The n-6-PUFA linoleic acid accounts for up to 18% of all FAs contained in multi-compendial grade PS80. 4-Hydroxynonenal (HNE) is highly reactive towards nucleophilic amino acids, potentially leading to covalent protein modifications. This study tests whether HNE may be a pharmaceutically relevant PS80 peroxidation product. Since HNE was not directly detectable in the PS80 matrix by UV and MS, a new quantification method was established. After derivatization with 2,4-dinitrophenyl hydrazine (DNPH) and extraction of the formed hydrazone with a salting-out assisted liquid-liquid extraction, the HNE-DNPH adduct was analyzed by multiple reaction monitoring. Kinetic oxidation studies were conducted incubating PS80 in presence and absence of the antioxidant butylhydroxytoluene (BHT). HNE was confirmed as PS80 degradant in oxidatively stressed samples. BHT was shown to prevent its formation. HNE is a detectable PS80 degradation product raising questions about the potential impact on critical quality attributes of biopharmaceuticals formulated with PS80. Addition of BHT prevented HNE formation under oxidative stress. Consequently, BHT might be a valuable additive in PS used in biopharmaceuticals.

Proteome-wide profiling and mapping of post translational modifications in human hearts

Post translational modifications (PTMs) are covalent modifications of proteins that can range from small chemical modifications to addition of entire proteins. PTMs contribute to regulation of protein function and thereby greatly increase the functional diversity of the proteome. In the heart, a few well-studied PTMs, such as phosphorylation and glycosylation, are known to play essential roles for cardiac function. Yet, only a fraction of the ~ 300 known PTMs have been studied in a cardiac context. Here we investigated the proteome-wide map of PTMs present in human hearts by utilizing high-resolution mass spectrometry measurements and a suite of PTM identification algorithms. Our approach led to identification of more than 150 different PTMs across three of the chambers in human hearts. This finding underscores that decoration of cardiac proteins by PTMs is much more diverse than hitherto appreciated and provides insights in cardiac protein PTMs not yet studied. The results presented serve as a catalogue of which PTMs are present in human hearts and outlines the particular protein and the specific amino acid modified, and thereby provides a detail-rich resource for exploring protein modifications in human hearts beyond the most studied PTMs.

Cooperative stabilisation of 14-3-3σ protein-protein interactions via covalent protein modification.

14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes via a large network of interactions with partner proteins. Many of these protein–protein interactions (PPI) are implicated in human diseases such as cancer and neurodegeneration. The stabilisation of selected 14-3-3 PPIs using drug-like ‘molecular glues’ is a novel therapeutic strategy with high potential. However, the examples reported to date have a number of drawbacks in terms of selectivity and potency. Here, we report that WR-1065, the active species of the approved drug amifostine, covalently modifies 14-3-3σ at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3σ PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3σ, an isoform with particular involvement in cancer pathways. This mechanism can be exploited to harness the enhanced potency conveyed by covalent drug molecules and dual ligand cooperativity. This is demonstrated in two cancer cell lines whereby the cooperative behaviour of fusicoccin A and WR-1065 leads to enhanced efficacy for inducing cell death and attenuating cell growth.

Bacterial Protein Kinases.

Bacteria are able to inhabit and survive vastly diverse environments. This enormous adaptive capacity depend on their ability to perceive cues from the micro-environment and process this information accordingly to mount appropriate metabolic responses and ultimately sustain homeostasis. From systems perspective, microbial cells conceal significant degree of organismal complexity, which may only be managed by continuous bulk cellular information flow and processing, inside the cell, between other cells and the environment. In this respect, reversible covalent modification of proteins is one of the universal mode of information flow mechanism used to regulate metabolism in all organisms. More than 30 types of post translational modifications have been identified, where phosphorylation constitutes nearly half of them. Bacterial cells possess several modes of phosphoprotein mediated information flow mechanisms. Histidine kinases and two component systems, bacterial tyrosine kinases, Hanks type serine/threonine kinases, atypical serine kinases and arginine kinases have been identified in many species.

Covalent Modification of Proteins by Plant-Derived Natural Products: Proteomic Approaches and Biological Impacts.

Plant-derived natural products (NPs) with electrophilic functional groups engage various subsets of the proteome via covalent modification of nucleophilic cysteine residues. This electrophile-nucleophile interaction can change protein conformation, alter protein function, and modulate their biological action. The biological significance of these covalent protein modifications in health and disease is increasingly recognized. One way to understand covalent NP-protein interactions is to utilize traditional proteomics and modern mass spectrometry (MS)-based proteomic strategies. These strategies have proven effective in uncovering specific NP protein targets and are critical first steps that allow for a much deeper understanding of the ability of NPs to modulate cellular processes. Here, plant-derived NPs that covalently modify proteins are reviewed, the biological significance of these covalent modifications, and the different proteomic strategies that have been employed to study these NP-protein interactions.

Characterization of the Class I MHC Peptidome Resulting From DNCB Exposure of HaCaT Cells.

Skin sensitization following the covalent modification of proteins by low molecular weight chemicals (haptenation) is mediated by cytotoxic T lymphocyte (CTL) recognition of human leukocyte antigen (HLA) molecules presented on the surface of almost all nucleated cells. There exist 3 nonmutually exclusive hypotheses for how haptens mediate CTL recognition: direct stimulation by haptenated peptides, hapten modification of HLA leading to an altered HLA-peptide repertoire, or a hapten altered proteome leading to an altered HLA-peptide repertoire. To shed light on the mechanism underpinning skin sensitization, we set out to utilize proteomic analysis of keratinocyte presented antigens following exposure to 2,4-dinitrochlorobenzene (DNCB). We show that the following DNCB exposure, cultured keratinocytes present cysteine haptenated (dinitrophenylated) peptides in multiple HLA molecules. In addition, we find that one of the DNCB modified peptides derives from the active site of cytosolic glutathione-S transferase-. These results support the current view that a key mechanism of skin sensitization is stimulation of CTLs by haptenated peptides. Data are available via ProteomeXchange with identifier PXD021373.

Covalent chemical modification of myofibrillar proteins to improve their gelation properties: A systematic review.

To improve the quality of meat products is a constant focus for both the meat industry and scientists. As major components in meat protein, the gelation properties of myofibrillar proteins (MPs) predominantly determine the sensory quality and product yield of the final product. Naturally or artificially occurring covalent modifications are known to largely affect MP functionality by changing the protein structure and forming aggregates, leading to both favorable and unfavorable outcomes. The review aims to summarize the mechanisms associated with several covalent modifications and the recent developments in enhancing MP gelation properties. Various extrinsic and intrinsic parameters controlling oxidation, phenolic-protein interactions, enzyme catalysis, glycation, and isoelectric solubilization/precipitation, and their effects on the characteristics of heat-induced MP gels are discussed. This article provides an improved understanding of the covalent modifications that occur mainly in the MP system and how they can be utilized to promote its gelation properties. Covalent modifications exhibited dose-dependent and dual-role manners for MP gelation properties. Mild oxidation, enzyme catalysis, and isoelectric solubilization/precipitation treatment would be beneficial to form more aligned and cross-linked three-dimensional networks for MP gels because of moderate protein aggregation. However, an excessive aggregate impedes the MP gelation behavior, leading to reduced gelation quality. Glycation effectively increased hydrophilicity of MPs and phenolic conjugation provides MPs with novel bioactivity. A proper utilization of such a process or even a rational combination of them allowed us to enhance the gelation properties of MP with assorted appreciated functionalities and further improve the quality of meat products.

Analysis of disulphide bond linkage between CoA and protein cysteine thiols during sporulation and in spores of Bacillus species.

ABSTRACTSpores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores’ metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes.

The Emerging Therapeutic Potential of Nitro Fatty Acids and Other Michael Acceptor-Containing Drugs for the Treatment of Inflammation and Cancer.

Nitro fatty acids (NFAs) are endogenously generated lipid mediators deriving from reactions of unsaturated electrophilic fatty acids with reactive nitrogen species. Furthermore, Mediterranean diets can be a source of NFA. These highly electrophilic fatty acids can undergo Michael addition reaction with cysteine residues, leading to post-translational modifications (PTM) of selected regulatory proteins. Such modifications are capable of changing target protein function during cell signaling or in biosynthetic pathways. NFA target proteins include the peroxisome proliferator-activated receptor γ (PPAR-γ), the pro-inflammatory and tumorigenic nuclear factor-κB (NF-κB) signaling pathway, the pro-inflammatory 5-lipoxygenases (5-LO) biosynthesis pathway as well as soluble epoxide hydrolase (sEH), which is essentially involved in the regulation of vascular tone. In several animal models of inflammation and cancer, the therapeutic efficacy of well-tolerated NFA has been demonstrated. This has already led to clinical phase II studies investigating possible therapeutic effects of NFA in subjects with pulmonary arterial hypertension. Albeit Michael acceptors feature a broad spectrum of bioactivity, they have for a rather long time been avoided as drug candidates owing to their presumed unselective reactivity and toxicity. However, targeted covalent modification of regulatory proteins by Michael acceptors became recognized as a promising approach to drug discovery with the recent FDA approvals of the cancer therapeutics, afatanib (2013), ibrutinib (2013), and osimertinib (2015). Furthermore, the Michael acceptor, neratinib, a dual inhibitor of the human epidermal growth factor receptor 2 and epidermal growth factor receptor, was recently approved by the FDA (2017) and by the EMA (2018) for the treatment of breast cancer. Finally, a number of further Michael acceptor drug candidates are currently under clinical investigation for pharmacotherapy of inflammation and cancer. In this review, we focus on the pharmacology of NFA and other Michael acceptor drugs, summarizing their potential as an emerging class of future antiphlogistics and adjuvant in tumor therapeutics.