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Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of protein targets for peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted LC-MS-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials.ImportanceThe frontline treatments for malaria are combination therapies based on the peroxide antimalarial, artemisinin. Concerningly, artemisinin resistance has emerged in malaria-endemic regions, and now poses a major threat to malaria treatment and eradication efforts. New medicines are urgently required to replace the artemisinins, and some of the most advanced candidates are the fully synthetic peroxide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel). The mechanism of action of peroxide antimalarials involves the reductive activation of the peroxide bond by intra-parasitic haem, but there is no consensus regarding the specific protein targets of the resulting radical species for artemisinins and/or the ozonides. This study provides a comprehensive and unbiased chemoproteomic profile of over 400 target proteins, and confirms the specific impact of peroxide antimalarials on redox metabolism. The key role of redox targets is particularly relevant considering that the mechanism of artemisinin resistance appears to involve modulation of peroxide activation and redox homeostasis.

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

Antimalarial peroxides such as the phytochemical artemisinin or the synthetic ozonides arterolane and artefenomel undergo reductive cleavage of the pharmacophoric peroxide bond by ferrous heme, released by parasite hemoglobin digestion. The generated carbon-centered radicals alkylate heme in an intramolecular reaction and proteins in an intermolecular reaction. Here, we determine the proteinaceous alkylation signatures of artemisinin and synthetic ozonides in Plasmodium falciparum using alkyne click chemistry probes to identify target proteins by affinity purification and mass spectrometry-based proteomics. Using stringent controls and purification procedures, we identified 25 P. falciparum proteins that were alkylated by the antimalarial peroxides in a peroxide-dependent manner, but the alkylation patterns were more random than we had anticipated. Moreover, there was little overlap in the alkylation signatures identified in this work and those disclosed in previous studies. Our findings suggest that alkylation of parasite proteins by antimalarial peroxides is likely to be a nonspecific, stochastic process.

In addition to supporting cellular energetic demands and providing building blocks for lipid synthesis, fatty acids (FAs) are precursors of potent signaling molecules. In particular, the presence of conjugated double bonds on the fatty-acyl chain provides a preferential target for nitration generating nitro-FAs (NO(2)-FAs). The formation of NO(2)-FAs is a nonenzymatic process that requires reactive nitrogen species and occurs locally at the site of inflammation or during gastric acidification. NO(2)-FAs are electrophilic and display pleiotropic signaling actions through reversible protein alkylation. This review focuses on the endogenously formed NO(2)-FAs, mechanism of absorption, systemic distribution, signaling, and preclinical models. Understanding the dynamics of these processes will facilitate targeted dietary interventions and further the current pharmacological development aimed at low-grade inflammatory diseases.

Identification of isomeric amino acid residues in peptides and proteins is challenging but often highly desired in proteomics. One of the practically important cases that require isomeric assignments is that associated with single-nucleotide polymorphism substitutions of Met residues by Thr in cancer-related proteins. These genetically encoded substitutions can yet be confused with the chemical modifications, arising from protein alkylation by iodoacetamide, which is commonly used in the standard procedure of sample preparation for proteomic analysis. Similar to the genetically encoded mutations, the alkylation also induces a conversion of methionine residues, but to the iso-threonine form. Recognition of the mutations therefore requires isoform-sensitive detection techniques. Herein, we demonstrate an analytical method for reliable identification of isoforms of threonine residues in tryptic peptides. It is based on ultraviolet photodissociation mass spectrometry of cryogenically cooled ions and a machine-learning algorithm. The measured photodissociation mass spectra exhibit isoform-specific patterns, which are independent of the residues adjacent to threonine or iso-threonine in a peptide sequence. A comprehensive metric-based evaluation demonstrates that, being calibrated with a set of model peptides, the method allows for isomeric identification of threonine residues in peptides of arbitrary sequence.

Se-benzyl selenoimidazolium salts are characterized by remarkable alkyl-transfer potential under physiological conditions. Structure-activity relationship studies show that selective monoalkylation of primary amines depends on supramolecular interactions between the selenoimidazole leaving group and the target nucleophile. We demonstrate that these reagents can be used for site-selective and nearly quantitative modification of the model protein lysozyme on Lys13, bypassing the higher intrinsic reactivities of Lys1 and Lys33. These observations introduce selenoimidazolium salts as novel class of electrophiles for selective N-alkylation of native proteins.

The incidence of papillary thyroid carcinomas (PTCs) has increased rapidly during the past several decades. Until now, the mechanisms underlying the tumorigenesis of PTCs have remained largely unknown. Next-generation-sequencing (NGS) provides new ways to investigate the molecular pathogenesis of PTCs. To characterize the somatic alterations associated with PTCs, we performed whole-exome sequencing (WES) of PTCs from 23 Chinese patients. This study revealed somatic mutations in genes with relevant functions for tumorigenesis, such as BRAF, BCR, CREB3L2, DNMT1, IRS2, MSH6, and TP53. We also identified novel somatic gene alterations which may be potentially involved in PTC progression. Gene set enrichment analysis revealed that the cellular response to hormone stimulus, epigenetic modifications, such as protein/histone methylation and protein alkylation, as well as MAPK, PI3K-AKT, and FoxO/mTOR signaling pathways, were significantly altered in the PTCs studied here. Moreover, Protein-Protein Interaction (PPI) network analysis of our mutated gene selection highlighted EP300, KRAS, PTEN, and TP53 as major core genes. The correlation between gene mutations and clinicopathologic features of the PTCs defined by conventional ultrasonography (US) and contrast-enhanced ultrasonography (CEUS) were assessed. These analyses established significant associations between subgroups of mutations and respectively taller-than-wide, calcified, and peak time iso- or hypo-enhanced and metastatic PTCs. In conclusion, our study supplements the genomic landscape of PTCs and identifies new actionable target candidates and clinicopathology-associated mutations. Extension of this study to larger cohorts will help define comprehensive genomic aberrations in PTCs and validate target candidates. These new targets may open methods of individualized treatments adapted to the clinicopathologic specifics of the patients.

We propose a new high-throughput ultrafast method for large-scale proteomics approaches by speeding up the classic filter aided sample preparation protocol, FASP, from overnight to 2.5 h. Thirty-six samples can be treated in 2.5 h, and the method is scalable to 96-well plate-based pipelines. After a modification of the FASP-tube, the steps of protein reduction, protein alkylation, and protein digestion of complex proteomes are done in just 5.25 min, each one under the effects of an ultrasonic field (7 cycles: 30 s on and 15 s off). The new method was compared to the standard overnight digestion FASP protocol, and no statistical differences were found for more than 92.4%, 92%, and 93.3% of the proteins identified by studying the proteome of E. coli, mouse brain, and mouse liver tissue samples, respectively. Furthermore, the successful relative label-free quantification of four spiked proteins in E. coli samples, BSA, β-lactoglobulin, α-casein, and α-lactalbumin, was achieved, using either the ultrasonic-based FASP protocol or the classic overnight one. The new US-FASP method matches the analytical minimalism rules as time, cost, sample requirement, reagent consumption, energy requirements, and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all of the advantages of the filter aided sample preparation protocol are maintained.

Staphylococcus aureus is a major human pathogen, which causes life-threatening systemic and chronic infections and rapidly acquires resistance to multiple antibiotics. Thus, new antimicrobial compounds are required to combat infections with drug resistant S. aureus isolates. The 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone lapachol was previously shown to exert antimicrobial effects. In this study, we investigated the antimicrobial mode of action of lapachol in S. aureus using RNAseq transcriptomics, redox biosensor measurements, S-bacillithiolation assays and phenotype analyses of mutants. In the RNA-seq transcriptome, lapachol caused an oxidative and quinone stress response as well as protein damage as revealed by induction of the PerR, HypR, QsrR, MhqR, CtsR and HrcA regulons. Lapachol treatment further resulted in up-regulation of the SigB and GraRS regulons, which is indicative for cell wall and general stress responses. The redox-cycling mode of action of lapachol was supported by an elevated bacillithiol (BSH) redox potential (EBSH), higher endogenous ROS levels, a faster H2O2 detoxification capacity and increased thiol-oxidation of GapDH and the HypR repressor in vivo. The ROS scavenger N-acetyl cysteine and microaerophilic growth conditions improved the survival of lapachol-treated S. aureus cells. Phenotype analyses revealed an involvement of the catalase KatA and the Brx/BSH/YpdA pathway in protection against lapachol-induced ROS-formation in S. aureus. However, no evidence for irreversible protein alkylation and aggregation was found in lapachol-treated S. aureus cells. Thus, the antimicrobial mode of action of lapachol in S. aureus is mainly caused by ROS formation resulting in an oxidative stress response, an oxidative shift of the EBSH and increased protein thiol-oxidation. As ROS-generating compound, lapachol is an attractive alternative antimicrobial to combat multi-resistant S. aureus isolates. Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

Alkyl halides are potentially mutagenic carcinogens. However, no efficient fluorescent sensor for alkyl halide detection in human-derived samples has been developed to date. Herein, we report a new protein-based fluorescent sensor for alkyl halides. Analysis of the HaloTag holo-crystal structure with its covalently attached ligand revealed an unexpected cavity, allowing for the design of a new fluorogenic ligand. This ligand showed the highest fluorescence response (300-fold) and fastest binding kinetics (t1/2 < 150 s) to a HaloTag mutant (M175P) protein. This protein-based sensor system was effectively used to detect alkyl halides in human serum and monitor real-time protein alkylation.

The photophysical properties and photochemical reactivities of a series of bis-naphthols 4a–4e and bis-anthrols 5a and 5e were investigated by preparative irradiation in CH3OH, fluorescence spectroscopy and laser flash photolysis (LFP). Methanolysis taking place via photodehydration (bis-naphthols: ΦR = 0.04–0.05) is in competition with symmetry breaking charge separation (SB-CS). The SB-CS gave rise to radical ions that were detected for 4a and 4e by LFP. Photodehydration gave quinone methides (QMs) that were also detected by LFP (λmax = 350 nm, τ ≈ 1–2 ms). In the aqueous solvent, excited state proton transfer (ESPT) competes with the abovementioned processes, giving rise to naphtholates, but the process is inefficient and can only be observed in the buffered aqueous solution at pH > 7. Since the dehydration of bis-naphthols delivers QMs, their potential antiproliferative activity was investigated by an MTT test on three human cancer cell lines (NCI-H1299, lung carcinoma; MCF-7, breast adenocarcinoma; and SUM159, pleomorphic breast carcinoma). Cells were treated with 4 or 5 with or without irradiation (350 nm). An enhancement of the activity (up to 10-fold) was observed upon irradiation, which may be associated with QM formation. However, these QMs do not cross-link DNA. The activity is most likely associated with the alkylation of proteins present in the cell cytoplasm, as evidenced by photoinduced alkylation of bovine and human serum albumins by 4a.

Many viruses, including the hepatitis C virus (HCV), are dependent on the host RNA silencing pathway for replication. In this study, we screened small molecule probes, previously reported to disrupt loading of the RNA-induced silencing complex (RISC), including 6-hydroxydopamine (6-OHDA), suramin (SUR), and aurintricarboxylic acid (ATA), to examine their effects on viral replication. We found that 6-OHDA inhibited HCV replication; however, 6-OHDA was a less potent inhibitor of RISC than either SUR or ATA. By generating a novel chemical probe (6-OHDA-yne), we determined that 6-OHDA covalently modifies host and virus proteins. Moreover, 6-OHDA was shown to be an alkylating agent that is capable of generating adducts with a number of enzymes involved in the oxidative stress response. Furthermore, modification of viral enzymes with 6-OHDA and 6-OHDA-yne was found to inhibit their enzymatic activity. Our findings suggest that 6-OHDA is a probe for oxidative stress as well as protein alkylation, and these properties together contribute to the antiviral effects of this compound.

In modern toxicology, substantial efforts are undertaken to develop alternative solutions for in vivo toxicity testing. The adverse outcome pathway (AOP) concept could facilitate knowledge-based safety assessment of chemicals that does not rely exclusively on in vivo toxicity testing. The construction of an AOP is based on understanding toxicological processes at different levels of biological organisation. Here, we present the developed AOP for liver fibrosis and demonstrate a linkage between hepatic injury caused by chemical protein alkylation and the formation of liver fibrosis, supported by coherent and consistent scientific data. This long-term process, in which inflammation, tissue destruction, and repair occur simultaneously, results from the complex interplay between various hepatic cell types, receptors, and signalling pathways. Due to the complexity of the process, an adequate liver fibrosis cell model for in vitro evaluation of a chemical’s fibrogenic potential is not yet available. Liver fibrosis poses an important human health issue that is also relevant for regulatory purposes. An AOP described with enough mechanistic detail might support chemical risk assessment by indicating early markers for downstream events and thus facilitating the development of an in vitro testing strategy. With this work, we demonstrate how the AOP framework can support the assembly and coherent display of distributed mechanistic information from the literature to support the use of alternative approaches for prediction of toxicity. This AOP was developed according to the guidance document on developing and assessing AOPs and its supplement, the users’ handbook, issued by the Organisation for Economic Co-operation and Development.

The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins alkylated by OZ277 and OZ439. To the best of the authors’ knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in P. falciparum.

Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellular context remain largely unexplored. We developed a new quantitative chemoproteomic platform, which uses isotopically tagged, photocleavable azido-biotin reagents to selectively capture and quantify the cellular targets labeled by the alkynyl analogue of HNE (aHNE). Our analyses site-specifically identified and quantified 398 aHNE protein alkylation events (386 cysteine sites and 12 histidine sites) in intact cells. This data set expands by at least an order of magnitude the number of such modification sites previously reported. Although adducts formed by Michael addition are thought to be largely irreversible, we found that most aHNE modifications are lost rapidly in situ. Moreover, aHNE adduct turnover occurs only in intact cells and loss rates are site-selective. This quantitative chemoproteomics platform provides a versatile general approach to map bioorthogonal-chemically engineered post-translational modifications and their cellular dynamics in a site-specific and unbiased manner.

Protein and DNA alkylation by endogenously produced electrophiles is associated with the pathogenesis of neurodegenerative diseases, to epigenetic alterations and to cell signaling and redox regulation. With the goal of visualizing, in real-time, the spatiotemporal response of the cell milieu to electrophiles, we have designed a fluorogenic BODIPY-acrolein probe, AcroB, that undergoes a350-fold fluorescence intensity enhancement concomitant with protein adduct formation. AcroB enables a direct quantification of single post-translational modifications occurring on cellular proteins via recording fluorescence bursts in live-cell imaging studies. In combination with super-resolution imaging, protein alkylation events may be registered and individually counted, yielding a map of protein-electrophile reactions within the cell lipid milieu. Alkylation is predominantly observed within mitochondria, a source, yet not a sink, of AcroB adducts, illustrating that a mitochondrial constitutive excretion mechanism ensures rapid disposal of compromised proteins. Sorting within the Golgi apparatus and trafficking along microtubules and through the cell endo- and exocytic pathways can be next visualized via live-cell imaging. Our results offer a direct visualization of cellular response to a noncanonical acrolein warhead. We envision AcroB will enable new approaches for diagnosis of pathologies associated with defective cellular trafficking. AcroB may help elucidate key aspects of mitochondria electrophile adduct excretion and cell endocytic and exocytic pathways. Conceptually, AcroB provides a new paradigm on fluorescence microscopy studies where chemical perturbation is achieved and simultaneously reported by the probe.

Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions.

Despite the fact that multiple artemisinin-alkylated proteins in Plasmodium falciparum have been identified in recent studies, the alkylation mechanism and accurate binding site of artemisinin-protein interaction have remained elusive. Here, we report the chemical-probe-based enrichment of the artemisinin-binding peptide and characterization of the artemisinin-binding site of P. falciparum translationally controlled tumor protein (TCTP). A peptide fragment within the N-terminal region of TCTP was enriched and found to be alkylated by an artemisinin-derived probe. MS2 fragments showed that artemisinin could alkylate multiple amino acids from Phe12 to Tyr22 of TCTP, which was supported by labeling experiments upon site-directed mutagenesis and computational modeling studies. Taken together, the "capture-and-release" strategy affords consolidated advantages previously unavailable in artemisinin-protein binding site studies, and our results deepened the understanding of the mechanism of protein alkylation via heme-activated artemisinin.

Liver fibrosis is an important human health issue associated with chemical exposure. It is a typical result of chronic toxic injury and one of the considered endpoints for regulatory purposes. This AOP describes the linkage between hepatic injury caused by protein alkylation and the formation of liver fibrosis. Fibrogenesis is a long-term and complex process for which an adequate cell model is not available and an in vitro evaluation of fibrogenic potential is therefore not feasible yet. This systematic and coherent display of currently available mechanistic-toxicological information can serve as a knowledge-based repository for identification/selection/development of in vitro methods suitable for measuring key events and their relationships along the AOP and to facilitate the use of alternative data for regulatory purposes. Identified uncertainties and knowledge gaps can indicate priorities for future research.

Background Alkylated DNA-protein alkyltransferases (AGTs) are conserved proteins that repair alkylation damage in DNA by using a single-step mechanism leading to irreversible alkylation of the catalytic cysteine in the active site. Trans-alkylation induces inactivation and destabilization of the protein, both in vitro and in vivo, likely triggering conformational changes. A complete picture of structural rearrangements occurring during the reaction cycle is missing, despite considerable interest raised by the peculiarity of AGT reaction, and the contribution of a functional AGT in limiting the efficacy of chemotherapy with alkylating drugs. Methods As a model for AGTs we have used a thermostable ortholog from the archaeon Sulfolobus solfataricus (SsOGT), performing biochemical, structural, molecular dynamics and in silico analysis of ligand-free, DNA-bound and mutated versions of the protein. Results Conformational changes occurring during lesion recognition and after the reaction, allowed us to identify a novel interaction network contributing to SsOGT stability, which is perturbed when a bulky adduct between the catalytic cysteine and the alkyl group is formed, a mandatory step toward the permanent protein alkylation. Conclusions Our data highlighted conformational changes and perturbation of intramolecular interaction occurring during lesion recognition and catalysis, confirming our previous hypothesis that coordination between the N- and C-terminal domains of SsOGT is important for protein activity and stability. General significance A general model of structural rearrangements occurring during the reaction cycle of AGTs is proposed. If confirmed, this model might be a starting point to design strategies to modulate AGT activity in therapeutic settings.

Sulfur mustard (SM) is a highly reactive compound that was discovered in the 19th century and has repeatedly been used as a chemical warfare agent. Despite decades of medical research, no causative treatment against SM exists. This is largely due to the highly complex pathophysiology at the cellular and intercellular level. This chapter describes the clinical symptoms with particular regard to the skin, eyes and respiratory systems. SM exerts its toxicity both through its primary effect, i.e. DNA and protein alkylation, and secondary mechanisms, including excessive inflammation, formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and poly(ADP-ribose) polymerase (PARP) overactivation. Loss of the phagocytotic ability of immune cells reduces the clearance of apoptotic bodies and necrotic cell debris. Cell migration is also impaired, negatively affecting immune cells and early endothelial cells, with adverse effects on wound healing. Regarding medical countermeasures against SM injury, this chapter outlines symptomatic therapy and describes the specific requirements and strategies in the search for novel antidotes. Corresponding to mechanisms of SM toxicity, current and potential therapeutic interventions are discussed, including direct scavenging of SM, anti-oxidative and anti-inflammatory therapy, limitation of extrinsic apoptosis and, theoretically, PARP inhibition. Some of the—currently most promising—compounds to treat SM injuries counteract multiple aspects of its toxicity: silibinin provides membrane stabilization, limits radical formation and enhances protein biosynthesis, whereas macrolide antibiotics limit necrosis and inflammation, and restore migratory and phagocytotic abilities of immune cells. For some compounds, including phytopharmaceuticals, no precise mechanism of action is currently known. Cell therapies are in their early experimental stages, but some approaches have been highly successful in animal studies and grafts of limbal corneal stem cells have even proven their clinical efficacy to counteract severe, long term effects of SM exposure. In summary, the current treatment of SM toxicity is mostly reliant on symptomatic interventions. A better understanding of its pathophysiology is likely to result in therapeutic advances in the near to medium term future. Pharmacological and cell therapeutic interventions both have a role in providing the best possible therapy for victims of SM exposure.

Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA alkyltransferases. These are proteins conserved in eucarya, bacteria and archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation. We report here in vitro and in vivo studies on the DNA alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. In addition, transcriptional and post-translational regulation of SsOGT by DNA damage was studied. We show that although the gene transcription is induced by alkylating agent treatment, the protein is degraded in vivo by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability.

Mitochondria play a key role in intracellular energy-generating processes, cell life and death, and are heavily involved in several metabolic pathways by integrating signaling networks; thus, a very large number of conditions are characterized by mitochondrial bioenergetic in humans. Often, mitochondrial changes are directly or indirectly dependent on the activation of intracellular stress cascades or death receptor-mediated pathways. Reactive oxygen species (ROS) formation, glutathione (GSH) depletion, protein alkylation and respiratory complex alterations are major events associated with mitochondrial dysfunction and represent critical initiating events in most forms of chronic liver disease. Through creating an analogy with a disrupted electric circuit gone bad, the present review focuses initially on how hepatic mitochondrial bioenergetics is affected in the context of drug and disease-induced liver failure and how targeting mitochondria with several antioxidant agents can be helpful for preventing the disruption of the mitochondrial electric circuit.

The current paper presents the synthesis and evaluation of a series of maleimides that were designed to inhibit the Cdc25 phosphatase by alkylation of catalytically essential cysteine residues. Although in HepB3 cell culture assays the analogues did exhibit antiproliferative IC(50) values ranging from sub-micromolar to greater than 100 microM, inhibition of Cdc25 through cysteine alkylation could not be demonstrated. It was also found that analysis using fluorescence activated cell sorting (FACS) following treatment with the most potent analogue (1t) did not provide data consistent with inhibition at one specific point in the cell cycle, as would be expected if Cdc25A were inhibited. Further studies with a subset of analogues resulted in a correlation of antiproliferative potencies with activation of the unfolded protein response (UPR). The UPR is a regulatory pathway that temporarily suspends protein production when misfolding of proteins occurs within the endoplastic reticulum (ER). In addition, ER chaperones that promote proper refolding become up-regulated. If cellular damage cannot be resolved by these mechanisms, then the UPR can initiate apoptosis. The current study indicates that these maleimide analogues lead to UPR activation, which is predictive of the selective antiproliferative activity of the series.

A new clean fast (8 min) method for in-solution protein digestion without detergent or urea for protein identification by peptide mass fingerprint and mass spectrometry-based techniques is proposed. The new method avoids the use of time consuming desalting procedures entailing the following four steps done under the effect of an ultrasonic field provided by a sonoreactor: denaturation (1 min) in a mixed solution of water:acetonitrile 1/1 (v/v); protein reduction (1 min); protein alkylation (1 min); and protein digestion (5 min). Five proteins with masses comprised between 14.4 kDa and 97 kDa and the protein split-soret cytochrome c from D. desulfuricans ATCC27774, were successfully identified with this procedure. No differences were found in the sequence coverage or in the number of peptides matched when the new clean method was compared to another one using urea. Twofold better signal-to-noise ratios were obtained in the MALDI spectra from protein samples prepared with the new method when comparing it with a method using urea. The new digestion method avoids the need to remove salt content and increases throughput (six samples at once) while reducing sample loss and contamination from sample handling.

Although some toxicological mechanisms of sulfur mustard (HD) have been uncovered, new knowledge will allow for advanced insight in the pathways that lead towards epidermal–dermal separation in skin. In the present investigation, we aimed to survey events that occur at the protein level in human epidermal keratinocytes (HEK) during 24 h after exposure to HD. By using radiolabeled 14C-HD, it was found that proteins in cultured HEK are significant targets for alkylation by HD. HD-adducted proteins were visualized by two-dimensional gel electrophoresis and analyzed by mass spectrometry. Several type I and II cytokeratins, actin, stratifin (14-3-3σ) and galectin-7 were identified. These proteins are involved in the maintenance of the cellular cytoskeleton. Their alkylation may cause changes in the cellular architecture and, in direct line with that, be determinative for the onset of vesication. Furthermore, differential proteomic analysis was applied to search for novel features of the cellular response to HD. Partial breakdown of type I cytokeratins K14, K16 and K17 as well as the emergence of new charge variants of the proteins heat shock protein 27 and ribosomal protein P0 were observed. Studies with caspase inhibitors showed that caspase-6 is probably responsible for the breakdown of type I cytokeratins in HEK. The significance of the results is discussed in terms of toxicological relevance and possible clues for therapeutic intervention.

Background and purpose: Phenyl-chloroethyl ureas (CEUs) are a class of anticancer drugs that mainly react with proteins. Two molecules of this family, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) induced G1/S and G2/M cell cycle blocks, respectively. We hypothesised that these observations were linked to a differential protein alkylation pattern. Experimental approach: Proteins from B16 cells incubated with [14C-urea]-CCEU and [125I]-ICEU were compared by 2D-analyses followed by MALDI-TOF identification of modified proteins and characterisation of the CCEU binding. Protein expression was investigated by Western blot analyses and cell cycle data were obtained by flow cytometry. Key results: Several proteins (PDIA1, PDIA3, PDIA6, TRX, VDAC2) were alkylated by both ICEU and CCEU but β-tubulin and prohibitin (PHB) were specifically alkylated by either ICEU or CCEU respectively. Specific alkylation of these two proteins might explain the observed difference in B16 cell cycle arrest in G2 and G1 phases respectively. Mass spectrometry studies on the alkylated prohibitin localised the modified peptide and identified Asp-40 as the target for CCEU. This alkylation induced an increased cellular content of PHB that should contribute to the accumulation of cells in G1 phase. Conclusions and implications: This study reinforces our findings that CEUs alkylate proteins through an ester linkage with an acidic amino acid and shows that PHB alkylation contributes to G1/S arrest in CCEU treated B16 cells. Modification of PHB status and/or activity is an open route for new cancer therapeutics. British Journal of Pharmacology (2007) 152, 449–455; doi:10.1038/sj.bjp.0707415; published online 20 August 2007

An ultrasonic bath, an ultrasonic probe and a sonoreactor were used to speed up the kinetics of the reactions involved in each step of the sample handling for in-gel protein identification by peptide mass fingerprint, PMF, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The following steps were successfully accelerated using ultrasonic energy: gel washing, protein reduction, and protein alkylation. As a result, a reduction comprising 80% to 90% of the total time involved in the classic approach was achieved. In addition the sample handling was also drastically simplified. The number of peptides identified and the protein sequence coverage obtained for the new procedure were comparable to those obtained with the traditional sample treatment for the following protein standards: glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and α-lactalbumin. Finally, as a proof of the procedure, specific proteins were identified from complex protein mixtures obtained from three different sulphate-reducing bacteria: Desulfovibrio desulfuricans G20, Desulfuvibrio gigas NCIB 9332, and Desulfuvibrio desulfuricans ATCC 27774. Copyright © 2007 John Wiley & Sons, Ltd.

Six monofunctional alkylating methanesulphonates of widely varying structures were investigated in the in vitro micronucleus assay with Syrian hamster embryo fibroblast cells. The results were compared with the alkylating activities measured in the 4-(nitrobenzyl)pyridine test (NBP-test) and the N -methyl mercaptoimidazole (MMI-test) as measures for S N 2 reactivity as well as in the triflouoroacetic acid (TFA) solvolysis and the hydrolysis reaction as measures for S N 1 reactivity in order to provide insights into the role of alkylation mechanisms on induction of micronuclei. Moreover we compared the results of micronucleus assay with those of the Ames tests in strain TA 100 and TA1535 and with those of the SOS chromotest with the strains PQ37, PQ243, PM21 and GC 4798. The potency of methanesulphonates to induce micronuclei depended only to a certain degree, on the total alkylating activity (S N 1 and S N 2 reactivity). An inverse, significant correlation between the Ames test and the micronucleus assay was observed and an inverse correlation between the micronucleus assay and the SOS chromotest with the different strains. The results indicate that the primary mechanism leading to induction of micronuclei is not O -alkylation in DNA as it is the case in the Ames test with the his G46 strains TA1535 and TA100 and not N -alkylation as with the SOS chromotest. There is evidence that protein alkylation, e.g. in the spindle apparatus in mitosis is decisive for induction of micronuclei by alkylating compounds. The structurally voluminous methanesulphonates 2-phenyl ethyl methanesulphonate and 1-phenyl-2-propyl methanesulphonate show a clear higher micronuclei inducing potency than the other tested though the bulky methanesulphonates possess a lower total alkylating activity than the others. This effect can be explained by a higher disturbance during mitosis after alkylation of the spindle apparatus with the structurally more bulky methanesulphonates.

O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs pro-mutagenic O(6)-alkylguanine and O(4)-alkylthymine lesions in DNA. The alkylated form of the protein is not reactivated; instead, it is rapidly ubiquitinated and degraded. Here, we show that alkylation destabilizes the native fold of the protein by 0.5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination.

S -Palmitoylation is a dynamic post-translational modification of certain proteins, which helps determine membrane association and may function to enhance the interactions of signaling molecules with their activated receptors and effector systems. Unlike enzymes that catalyze other protein lipidation reactions, e.g. farnesylation and N -myristoylation, protein palmitoyltransferase is virtually uncharacterized biochemically. We have described previously the synthesis of cerulenin analogs including cis -2,3-epoxy-4-oxononadecanamide (16C) and cis -2,3-epoxy-4-oxododecanamide (9C) that inhibit protein palmitoylation (Lawrence et al., J Med Chem 1999;42:4932–41), most likely through covalent alkylation of protein palmitoyltransferase. [ 3 H]9C and [ 3 H]16C were prepared by catalytic incorporation of 3 H 2 into unsaturated precursors for further study of their cellular pharmacology. After 4 hr, T24 bladder carcinoma cells in the absence of serum accumulated a 4-fold higher intracellular level of [ 3 H]16C than of [ 3 H]9C. Uptake of [ 3 H]9C and [ 3 H]16C was reduced by the presence of serum in the medium, suggesting their binding to serum proteins. [ 3 H]9C and [ 3 H]16C alkylated unique patterns of proteins in T24 cells, with proteins of approximately 80 and 31 kDa being labeled by each compound. A panel of human tumor cell lines demonstrated half-maximal proliferation inhibition at concentrations of 7–30, 4–16, and 8–36 μM, for cerulenin, 9C, and 16C, respectively, indicating that the cell lines have approximately equal sensitivity to these compounds. Different cell lines have similar patterns of protein alkylation by [ 3 H]9C or [ 3 H]16C, with labeling intensity related to cytotoxicity of the compounds. Since both 9C and 16C inhibit palmitoylation, the commonly labeled proteins are candidates for human protein palmitoyltransferase.

There are several known routes for the metabolic detoxication of α,β-unsaturated aldehydes and ketones, including conjugation to glutathione and reduction and oxidation of the aldehyde to an alcohol and a carboxylic acid, respectively. In this study, we describe a fourth class of detoxication that involves the reduction of the α,β-carbon=carbon double bond to a single bond. This reaction is catalyzed by NAD(P)H-dependent alkenal/one oxidoreductase (AO), an enzyme heretofore known as leukotriene B4 12-hydroxydehydrogenase, 15-oxoprostaglandin 13-reductase, and dithiolethione-inducible gene-1. AO is shown to effectively reduce cytotoxic lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) (k cat = 4.0 × 103 min−1;k cat/K m = 3.3 × 107 min−1 m −1) and acrolein (k cat = 2.2 × 102min−1; k cat/K m= 1.5 × 106 min−1 m −1) and common industrial compounds such as ethyl vinyl ketone (k cat = 9.6 × 103 min−1;k cat/K m = 8.8 × 107 min−1 m −1) and 15-oxoprostaglandin E1 (k cat = 2.4 × 103 min−1;k cat/K m = 2.4 × 109 min−1 m −1). Furthermore, transfection of human embryonic kidney cells with a rat liver AO expression vector protected these cells from challenge with HNE. The concentration of HNE at which 50% of the cells were killed after 24 h increased from ∼15 μm in control cells to ∼70 μm in AO-transfected cells. Overexpression of AO also completely abolished protein alkylation by HNE at all concentrations tested (up to 30 μm). Thus, we describe a novel antioxidative activity of a previously characterized bioactive lipid-metabolizing enzyme that could prove to be therapeutically or prophylactically useful due to its high catalytic rate and inducibility.

All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.

The present review highlights some important alkylation pathways of proteins, as measured by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometric analysis, engendered by acrylamide and a number of its derivatives, including N-substituted acrylamides, cross-linkers and Immobilines (the acrylamido weak acids and bases used to create immobilized pH gradients). The present data are of relevance in two-dimensional maps and proteome analysis. It is shown that acrylamide can alkylate the -SH group of proteins even when engaged in disulfide bridges. An order of reactivity is obtained for a series of cross-linkers, which are shown to have an extremely reacting double bond, with the second one almost unreactive, originating "pendant, unreacted ends", which can subtract proteins migrating in a gel by covalently affixing them to it. An analogous reactivity scale is constructed also for the Immobiline chemicals, whose reactivity is shown to be linearly dependent on the pK values, the least reacting species being the acidic compounds. When analyzing real-life samples by two-dimensional (2-D) maps, like milk powders, a number of modifications can be detected by MALDI-TOF mass spectra of eluted spots, including variable phosphorylation sites (up to nine) and lactosyl moieties. If, for eluting such spots, formic acid is used, MALDI-TOF mass spectrometry (MS) reveals an incredible number of formylation sites, on Ser and Thr residues.

A number of cross-linkers that are commonly used in polyacrylamide gels have been incubated with bovine beta-lactoglobulin B and the resulting reaction mixtures were examined by matrix assisted laser desorption/ionization-mass spectrometry. At concentrations of 0.1, 1, and 20 mM of each cross-linker incubated for 1 h with 50 pmol/microL of the protein, a reactivity scale can be expressed as polyethylene glycol diacrylate > N,N'-bisacrylylcystamine > bisacrylyl piperazine > N,N'-methylenebisacrylamide >> N,N'-diallyltartardiamide (PEGDA>BAC>BAP>Bis>>DATD). Relatively short incubation times indicated one of the five Cys residues as the target of reaction, which was confirmed by post-source decay measurements. Longer incubation times (24 h) with bisacrylamide extended the reaction to all five Cys residues and a number of Lys residues. A second consequence of longer reaction time is the involvement of both terminals of the cross-linker in the observed reaction. This experimental evidence is the first to demonstrate a different reactivity of both ends of one of the most commonly used cross-linkers. Investigation of solutions containing a cross-linker and acrylamide monomers provided useful information on the competition between the two identities for reaction with the protein. Possible implications of these experimental observations for isoelectric focusing separations in polyacrylamide gels are discussed.

A number of Immobilines, with pK 1.0-10.3, were incubated with two proteins, bovine alpha-lactalbumin (pI 4.80) and chicken egg lysozyme (pI 9.32), at pH approximately 9-10 and the resulting solutions were examined by matrix assisted laser desorption/ionization mass spectrometry. The reflectron mode of the same technique was also used to analyze a number of tryptic digests of some solutions. The extent and the number of detected alkylation sites associated with the acidic protein were found to be linearly proportional to the pK values of the investigated Immobilines, an effect which was less evident for the basic protein. The high resolution measurements of some tryptic digests indicate the cysteine residues as the likely sites of alkylation. The implications of the present data for isoelectric focusing separations on immobilized pH gradients and for two-dimensional maps are discussed.

In essential hypertension and diabetic nephropathy, sodium-lithium countertransport (Na-Li CT) is an inherited marker, subject to metabolic influences, of cardiovascular risk. Studies in Type II diabetes, taking clinical phenotypes as their starting point, are conflicting. We sought to identify Na-Li CT kinetic abnormalities in Type II diabetes, and only subsequently to seek relationships with clinical variables. Na-Li CT kinetics, membrane fluidity and their modulation by thiol proteins were measured in erythrocytes from 38 patients with Type II diabetes and in 16 normal control subjects. In untreated erythrocytes, Na-Li CT kinetics were similar. Thiol protein alkylation with N-ethylmaleimide generally caused both V max and K m to fall, but caused K m to rise in erythrocytes from 13 out of 38 diabetic subjects, whose native K m was low (P = 0.0013 compared with control). V max and serum triacylglycerol levels were related in normal controls (r s = 0.54, P = 0.038) and in diabetic subjects whose K m fell after N-ethylmaleimide (n = 25, r s = 0.62, P = 0.001). Where the K m rose after N-ethylmaleimide, V max and triacylglycerol levels were not related (n = 13, r s = -0.39, P = 0.183) and membrane fluidity did not increase after N-ethylmaleimide. However, these subgroups were indistinguishable in terms of blood pressure, albuminuria, glycaemia or lipid profiles. Thus abnormalities in the regulation of Na-Li CT and membrane fluidity by key thiol proteins, resembling those seen in essential hypertension and diabetic nephropathy, were apparent in one-third of subjects with Type II diabetes. Membrane abnormalities may indicate a common pathological mechanism. The prognostic significance of Na-Li CT kinetic abnormalities in Type II diabetes must now be confirmed.

4-Trifluoromethylphenol (4-TFMP) was cytotoxic to precision-cut rat liver slices as indicated by loss of intracellular potassium. Intracellular glutathione levels decreased and fluoride ion levels increased in a time and concentration-dependent manner. The cytotoxicity of 4-TFMP did not appear to be due to the release of fluoride, however, since equimolar concentrations of sodium fluoride or potassium fluoride were not toxic. The ortho isomer (2-TFMP), which had a threefold slower rate of fluoride release, was much less toxic to liver slices. In incubations without slices, 4-TFMP spontaneously hydrolyzed in aqueous buffer at physiological pH to form 4-hydroxybenzoic acid via a quinone methide intermediate. The quinone methide was trapped by the addition of glutathione. Analysis of the glutathione adduct indicated that all of the fluorine atoms were lost during the hydrolysis, yielding a cresol derivative with the glutathione moiety attached to a benzylic carbonyl group. The glutathione conjugate was the primary product formed at low alkylphenol/glutathione ratios; however, at higher 4-TFMP concentrations additional unidentified products were observed. 4-TFMP also inhibited the in vitro enzyme activity of purlfied glyceraldehyde-3-phosphate dehydrogenase, a sulfhydryl-dependent enzyme, in a time and concentration-dependentmanner. Loss of thiol residues closely paralleled the loss in enzyme activity. The coaddition of glutathione prevented 4-TFMP-induced loss of enzyme activity. The cytotoxicity of 4-TFMP therefore appears to be due to spontaneous quinone methide formation and subsequent alkylation of cellular macromolecules.

The recent development of several promising new thiourea-containing drugs has renewed interest in the thiourea functionality as a potential toxicophore. Most adverse reactions of thiourea-containing compounds are attributed to the thionocarbonyl moiety. Oxidation of these thionocarbonyl compounds by flavin-containing monooxygenases (FMO) and cytochrome P450 isoenzymes (P450) to reactive sulfenic, sulfinic, or sulfonic acids leads to alkylation of essential macromolecules. To more rationally design thiourea-containing drugs, structure-toxicity relationships (STRs) must be derived. Since for the development of STRs a large number of thiourea-containing compounds must be investigated, it is important to develop rapid in vitro assays for alkylating potential. In this study, the utility of activation of microsomal glutathione S-transferase (mGST) and inactivation of P450 1A1 as markers of the alkylating potential of metabolites of thiourea-containing compounds was investigated. It was found that metabolites of thiourea-containing compounds inactivate P450 1A1 in a time-dependent manner, as evidenced by a decrease in 7-ethoxyresorufin O-dealkylation (EROD) activity. An extent of inactivation of P450 1A1 by 100 microM N-phenylthiourea (PTU) of 64% was found after 10 min. This inactivation was dependent on the presence of NADPH and the presence of the thionosulfur, since the carbonyl analogue of PTU was not found to inactivate P450 1A1, and was partially prevented by heat treatment of the microsomes which is known to selectively inactivate FMO enzymes. Inactivation of P450 1A1 could be reversed by treatment with dithiothreitol, indicating the formation of disulfide bonds. However, thiourea-containing compounds also inhibited the EROD activity of P450 1A1 in a competitive manner. This property complicates the usefulness of the EROD activity of P450 1A1 as a marker for the alkylating potential of thiourea-containing compounds. It was found that metabolites of thiourea-containing compounds could transiently activate the mGST. A maximal level of activation by 100 microM PTU of 162+/-16% was found after 10 min. Activation of mGST by 100 microM PTU was dependent on the presence of NADPH and the presence of the thionosulfur, since the carbonyl analogue of PTU was not found to activate mGST. Activation was completely prevented by heat treatment of the microsomes, indicating involvement of FMO in the bioactivation process. Finally, a series of structurally diverse thiourea-containing compounds were tested for their ability to activate mGST. It appeared that their potency in alkylating mGST was inversely related to their Vmax/Km value for the FMO enzyme. From this study, it is concluded that, whereas activation of mGST in rat liver microsomes may be a useful system with which to investigate the relationship between structure and alkylating potential of thiourea-containing compounds in vitro, inactivation of P450 1A1 is not.