Mass Spectrometry-Based Strategies for Assessing Human Exposure Using Hemoglobin Adductomics.

Hemoglobin (Hb) adducts are widely used in human biomonitoring due to the high abundance of hemoglobin in human blood, its reactivity toward electrophiles, and adducted protein stability for up to 120 days. In the present paper, we compared three methods of analysis of hemoglobin adducts: mass spectrometry of derivatized N-terminal Val adducts, mass spectrometry of N-terminal adducted hemoglobin peptides, and limited proteolysis mass spectrometry . Blood from human donors was incubated with a selection of contact allergens and other electrophiles, after which hemoglobin was isolated and subjected to three analysis methods. We found that the FIRE method was able to detect and reliably quantify N-terminal adducts of acrylamide, acrylic acid, glycidic acid, and 2,3-epoxypropyl phenyl ether (PGE), but it was less efficient for 2-methyleneglutaronitrile (2-MGN) and failed to detect 1-chloro-2,4-dinitrobenzene (DNCB). By contrast, bottom-up proteomics was able to determine the presence of adducts from all six electrophiles at both the N-terminus and reactive hemoglobin side chains. Limited proteolysis mass spectrometry, studied for four contact allergens (three electrophiles and a metal salt), was able to determine the presence of covalent hemoglobin adducts with one of the three electrophiles (DNCB) and coordination complexation with the nickel salt. Together, these approaches represent complementary tools in the study of the hemoglobin adductome.

Investigation into Propolis Components Responsible for Inducing Skin Allergy: Air Oxidation of Caffeic Acid and Its Esters Contribute to Hapten Formation.

Propolis is a resin-like material produced by bees from the buds of poplar and cone-bearing trees and is used in beehive construction. Propolis is a common additive in various biocosmetics and health-related products, despite the fact that it is a well-known cause of contact allergy. Caffeic acid and its esters have been the primary suspects behind the sensitization potency of propolis-induced contact allergy. However, the chemical structures of the protein adducts formed between these haptens and skin proteins during the process of skin sensitization remain unknown. In this study, the reactivity of three main contact allergens found in propolis, namely, caffeic acid (CA), caffeic acid 1,1-dimethylallyl ester (CAAE), and caffeic acid phenethyl ester (CAPE), was investigated. These compounds were initially subjected to the kinetic direct peptide reactivity assay to categorize the sensitization potency of CA, CAAE, and CAPE, but the data obtained was deemed too unreliable to confidently classify their skin sensitization potential based on this assay alone. To further investigate the chemistry involved in generating possible skin allergy-inducing protein adducts, model peptide reactions with CA, CAAE, and CAPE were conducted and analyzed via liquid chromatography-high-resolution mass spectrometry. Reactions between CA, CAAE, and CAPE and a cysteine-containing peptide in the presence of oxygen, both in closed and open systems, were monitored at specific time points. These studies revealed the formation of two different adducts, one corresponding to thiol addition to the α,ß-unsaturated carbonyl region of the caffeic structure and the second corresponding to thiol addition to the catechol, after air oxidation to o-quinone. Observation of these peptide adducts classifies these compounds as prehaptens. Interestingly, no adduct formation was observed when the same reactions were performed under oxygen-free conditions, highlighting the importance of air oxidation processes in CA, CAAE, and CAPE adduct formation. Additionally, through NMR analysis, we found that thiol addition occurs at the C-2 position in the aromatic ring of the CA derivatives. Our results emphasize the importance of air oxidation in the sensitization potency of propolis and shed light on the chemical structures of the resultant haptens which could trigger allergic reactions in vivo.

Nature-Derived Epoxy Resin Monomers with Reduced Sensitizing Capacity─Isosorbide-Based Bis-Epoxides.

Epoxy resin systems (ERSs) are a class of thermosetting resins that become thermostable and insoluble polymers upon curing. They are widely used as components of protective surfaces, adhesives, and paints and in the manufacturing of composites in the plastics industry. The diglycidyl ether of bisphenol A (DGEBA) is used in 75-90% of ERSs and is thus by far the most used epoxy resin monomer (ERM). Unfortunately, DGEBA is a strong skin sensitizer and it is one of the most common causes of occupational contact dermatitis. Furthermore, DGEBA is synthesized from bisphenol A (BPA), which is a petroleum-derived chemical with endocrine-disruptive properties. In this work, we have used isosorbide, a renewable and nontoxic sugar-based material, as an alternative to BPA in the design of ERMs. Three different bis-epoxide isosorbide derivatives were synthesized: the diglycidyl ether of isosorbide (1) and two novel isosorbide-based bis-epoxides containing either a benzoic ester (2) or a benzyl ether linkage (3). Assessment of the in vivo sensitizing potency of the isosorbide bis-epoxides in the murine local lymph node assay (LLNA) showed that all three compounds were significantly less sensitizing than DGEBA, especially 2 which was nonsensitizing up to 25% w/v. The peptide reactivity showed the same order of reactivity as the LLNA, i.e., 2 being the least reactive, followed by 3 and then 1, which displayed similar peptide reactivity as DGEBA. Skin permeation of 2 and 3 was compared to DGEBA using ex vivo pig skin and static Franz cells. The preliminary investigations of the technical properties of the polymers formed from 1-3 were promising. Although further investigations of the technical properties are needed, all isosorbide bis-epoxides have the potential to be less sensitizing renewable replacements of DGEBA, especially 2 that had the lowest sensitizing potency in vivo as well as the lowest peptide reactivity.

Pathways to Identify Electrophiles In Vivo Using Hemoglobin Adducts: Hydroxypropanoic Acid Valine Adduct and Its Possible Precursors.

Analytical methods and tools for the characterization of the human exposome by untargeted mass spectrometry approaches are advancing rapidly. Adductomics methods have been developed for untargeted screening of short-lived electrophiles, in the form of adducts to proteins or DNA, in vivo. The identification of an adduct and its precursor electrophile in the blood is more complex than that of stable chemicals. The present work aims to illustrate procedures for the identification of an adduct to N-terminal valine in hemoglobin detected with adductomics, and pathways for the tracing of its precursor and possible exposure sources. Identification of the adduct proceeded via preparation and characterization of standards of adduct analytes. Possible precursor(s) and exposure sources were investigated by measurements in blood of adduct formation by precursors in vitro and adduct levels in vivo. The adduct was identified as hydroxypropanoic acid valine (HPA-Val) by verification with a synthesized reference. The HPA-Val was measured together with other adducts (from acrylamide, glycidamide, glycidol, and acrylic acid) in human blood (n = 51, schoolchildren). The HPA-Val levels ranged between 6 and 76 pmol/g hemoglobin. The analysis of reference samples from humans and rodents showed that the HPA-Val adduct was observed in all studied samples. No correlation of the HPA-Val level with the other studied adducts was observed in humans, nor was an increase in tobacco smokers observed. A small increase was observed in rodents exposed to glycidol. The formation of the HPA-Val adduct upon incubation of blood with glycidic acid (an epoxide) was shown. The relatively high adduct levels observed in vivo in relation to the measured reactivity of the epoxide, and the fact that the epoxide is not described as naturally occurring, suggest that glycidic acid is not the only precursor of the HPA-Val adduct identified in vivo. Another endogenous electrophile is suspected to contribute to the in vivo HPA-Val adduct level.

Haptenation of Macrophage Migration Inhibitory Factor: A Potential Biomarker for Contact Hypersensitivity.

The immunological response in contact hypersensitivity is incited by small electrophilic compounds, known as haptens, that react with endogenous proteins after skin absorption. However, the identity of hapten-modified proteins seen as immunogenic remains as yet largely unknown. In a recent study, we have for the first time identified a hapten-modified protein in the local lymph nodes of mice treated topically with the model hapten tetramethylrhodamine isothiocyanate (TRITC). The TRITC modification was located on the N-terminal proline of the protein macrophage migration inhibitory factor (MIF). The focus of the current study was to investigate the presence of the same hapten-protein conjugate in blood samples from mice treated topically with TRITC. Furthermore, TRITC modifications of the two major blood proteins, namely hemoglobin (Hb) and albumin (Alb), as well as TRITC modifications of MIF other than the N-terminal proline, were examined. Following incubation with different molar ratios of TRITC, a proteomic approach was applied to characterize conjugate formation of the three aforementioned proteins, using high resolution mass spectrometry (HRMS). The targeted screening of the TRITC-treated mice blood and lymph node samples for these sites led to the identification of only the same TRITC-MIF conjugate previously detected in the lymph nodes. No Hb and Alb conjugates were detected. Quantification of both the TRITC-modified and unmodified N-terminal peptide of MIF in blood and lymph node samples gave interesting insights of MIF's role in murine contact hypersensitivity. Incubation of MIF with four different haptens encompassing different reactivity mechanisms and potencies, showed adduct formation at different amino acid residues, suggesting that MIF can be the preferred target for a wide variety of haptens. The present study provides essential progress toward understanding of hapten-protein conjugate formation in contact hypersensitivity and identifies hapten-modified MIF as a potential biomarker for this condition. Further investigation of MIF as a target protein can be a next step to determine if MIF is a biomarker that can be used to develop better diagnostic tools and targeted therapeutics for individuals with allergic contact dermatitis.

Novel 4-Hydroxybenzyl Adducts in Human Hemoglobin: Structures and Mechanisms of Formation.

Humans are exposed to large numbers of electrophiles from their diet, the environment, and endogenous physiological processes. Adducts formed at the N-terminal valine of hemoglobin are often used as biomarkers of human exposure to electrophilic compounds. We previously reported the formation of hemoglobin N-terminal valine adducts (added mass, 106.042 Da) in the blood of human smokers and nonsmokers and identified their structure as 4-hydroxybenzyl-Val. In the present work, mass spectrometry-based proteomics was utilized to identify additional sites for 4-hydroxybenzyl adduct formation at internal nucleophilic amino acid side chains within hemoglobin. Hemoglobin isolated from human blood was treated with para-quinone methide (para-QM) followed by global nanoLC-MS/MS and targeted nanoLC-MS/MS to identify amino acid residues containing the 4-hydroxybenzyl modification. Our experiments revealed the formation of 4-hydroxybenzyl adducts at the αHis20, αTyr24, αTyr42, αHis45, ßSer72, ßThr84, ßThr87, ßSer89, ßHis92, ßCys93, ßCys112, ßThr123, and ßHis143 residues (in addition to N-terminal valine) through characteristic MS/MS spectra. These amino acid side chains had variable reactivity toward para-QM with αHis45, αTyr42, ßCys93, ßHis92, and ßSer72 forming the largest numbers of adducts upon exposure to para-QM. Two additional mechanisms for formation of 4-hydroxybenzyl adducts in humans were investigated: exposure to 4-hydroxybenzaldehyde (4-HBA) followed by reduction and UV-mediated reactions of hemoglobin with tyrosine. Exposure of hemoglobin to a 5-fold molar excess of 4-HBA followed by reduction with sodium cyanoborohydride produced 4-hydroxybenzyl adducts at several amino acid side chains of which αHis20, αTyr24, αTyr42, αHis45, ßSer44, ßThr84, and ßHis92 were verified in targeted mass spectrometry experiments. Similarly, exposure of human blood to ultraviolet radiation produced 4-hydroxybenzyl adducts at αHis20, αTyr24, αTyr42, αHis45, ßSer44, ßThr84, and ßSer89. Overall, our results reveal that 4-hydroxybenzyl adducts form at multiple nucleophilic sites of hemoglobin and that para-QM is the most likely source of these adducts in humans.

Characterizing Adduct Formation of Electrophilic Skin Allergens with Human Serum Albumin and Hemoglobin.

Skin (contact) allergy, the most predominant form of immunotoxicity in humans, is caused by small electrophilic compounds (haptens) that modify endogenous proteins. Approximately 20% of the general population in the Western world is affected by contact allergy. Although the importance of the hapten-protein conjugates is well established in the initiation of the immunological reaction, not much progress has been made regarding identification of these conjugates in vivo or exploration of their potential as diagnostic tools. In this study, the human serum albumin (HSA) and human hemoglobin (Hb) adductome for three representative contact allergens with different chemical properties, 1-chloro-2,4-dinitrobenzene (DNCB), 1,2-epoxy-3-phenoxypropane (PGE), and 2-bromo-2-(bromomethyl)glutaronitrile (MDBGN), were studied. Plasma and red blood cell lysate were used as a source for HSA and Hb, respectively. The Direct Peptide Reactivity Assay was used to investigate adduct formation of MDBGN with nucleophilic moieties and revealed that MDGBN is converted to 2-methylenepentanedinitrile in the presence of sulfhydryl groups prior to adduct formation. Following incubation of HSA and Hb with haptens, an Orbitrap Q Exactive high-resolution mass spectrometer was used to perform an initial untargeted analysis to screen for adduct formation, followed by confirmation by targeted Parallel Reaction Monitoring analysis. Although a subset of adducted sites was confirmed by targeted analysis, only some of the adducted peptides showed an increase in the relative amount of the adducted peptide with an increased concentration of hapten. In total, seven adduct sites for HSA and eight for Hb were confirmed for DNCB and PGE. These sites are believed to be the most reactive. Further, three of the HSA sites (Cys34, Cys62, and Lys190) and six of the Hb sites (subunit α: Val1, His45, His72; subunit ß: Cys93, His97, and Cys112) were haptenated already at the lowest level of hapten to protein molar ratio (0.1:1), indicating that these sites are the most likely to be modified in vivo. To the best of our knowledge, this is the first time that the adductome of Hb has been studied in the context of contact allergens. Identification of the most reactive sites of abundant proteins, such as HSA and Hb, is the first step toward identification of contact allergy biomarkers that can be used for biomonitoring and to develop better diagnostic tools based on a blood sample.

N-Glycosylation profiling of intact target proteins by high-resolution mass spectrometry (MS) and glycan analysis using ion mobility-MS/MS.

Glycosylation influences the structure and functionality of glycoproteins, and is regulated by genetic and environmental factors. The types and abundance of glycans on glycoproteins can vary due to diseases such as cancer, inflammation, autoimmune and neurodegenerative disorders. Due to the crucial role glycans play in modulating protein function, glycosylation analysis could lead to the discovery of novel biomarkers and is of prime importance in controlling the quality of glycoprotein biopharmaceuticals. Here, we present a method for the identification and quantification of glycoforms directly on intact proteins, after immunoaffinity purification from biological fluids. The method was validated and applied to serum transferrin and the biopharmaceutical trastuzumab. The accuracy of the method, expressed as the relative error (RE), ranged from 2.1 (at high concentrations) to 7.9% (at low concentrations), and intra- and inter-day precision, expressed as relative standard deviation (RSD), was 3.2 and 8.2%, respectively. The sensitivity and linearity of the method were suitable for serum analysis and the LOQ was calculated to be 3.1 and 4.4 µg mL-1 for transferrin (TFN) and trastuzumab (TRA), respectively. Its application to transferrin from five healthy human serum samples yielded concentrations between 1.61 and 3.17 mg mL-1, which are in agreement with blood reference levels. In parallel, the structure of the identified glycans was determined by ion mobility spectrometry coupled with tandem mass spectrometry. No chromatographic separation was required and sample preparation was performed in a semi-automatic manner, facilitating the handling of up to 12 samples at a time. This method should be useful for clinical laboratories and for the quality control of large batches of biopharmaceuticals.

Development of New Epoxy Resin Monomers - A Delicate Balance between Skin Allergy and Polymerization Properties.

Epoxy resin monomers (ERMs) are used as building blocks for thermosetting polymers in applications where strong, flexible, and lightweight materials are required. Most epoxy resins are polymers of diglycidyl ether of bisphenol A (DGEBA). It is highly allergenic and causes occupational allergic contact dermatitis and contact allergy in the general population. Thus, measures to prevent exposure by protective clothing and education are not enough. This work describes a continuation of our research aiming at reducing the skin-sensitizing potency of ERMs while maintaining the ability to form polymers. Alternative ERMs were designed and synthesized whereafter the sensitizing potency was determined using the murine local lymph node assay (LLNA). The reactivity of the diepoxides toward a nucleophilic peptide was investigated, and the differences in reactivity explained using computational studies. The diepoxides were reacted with triethylenetetramine, and the formed polymers were tested for technical applicability using thermogravimetric analysis. We had previously shown that the absence of an oxygen atom in the side chains or removal of aromaticity reduced the sensitizing potency compared to that of DGEBA. Thus, a cycloaliphatic analogue 1 of DGEBA without ether oxygen in the side chains was considered promising and was synthesized. As predicted, the sensitizing potency was considerably reduced (10 times) compared to that of DGEBA. However, the technical properties of the polymer of this compound were not considered sufficient. More polar aromatic analogues were investigated, but they could not compete with our previously described ERMs regarding polymerization properties and with 1 regarding low skin sensitization properties. Development of alternative epoxy materials is a delicate balance between allergenic activity and polymerization properties. Tuning of structural properties together with investigation of polymerization conditions combined with skin sensitization studies should be used in industrial research and development. ERM 1 could be used as a lead compound for further studies of aliphatic ERMs.

Discovery of Novel N-(4-Hydroxybenzyl)valine Hemoglobin Adducts in Human Blood.

Humans are exposed to a wide range of electrophilic compounds present in our diet and environment or formed endogenously as part of normal physiological processes. These electrophiles can modify nucleophilic sites of proteins and DNA to form covalent adducts. Recently, powerful untargeted adductomic approaches have been developed for systematic screening of these adducts in human blood. Our earlier untargeted adductomics study detected 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. We now describe a full characterization of one of these adducts, which corresponds to the addition of a 4-hydroxybenzyl (4-OHBn) group to N-terminal valine in Hb to form N(4-hydroxybenzyl)valine (4-OHBn-Val). The adduct structure was determined by comparison of its accurate mass, HPLC retention time, and MS/MS fragmentation to that of authentic standards prepared by chemical synthesis. Average 4-OHBn-Val adduct concentrations in 12 human blood samples were estimated to 380 ± 160 pmol/g Hb. Two possible routes of 4-OHBnVal adduct formation are proposed using two different precursor electrophiles: 4-quinone methide (4-QM) and 4-hydroxybenzaldehyde (4-OHBA). We found that 4-QM reacts rapidly with valine to form the 4-OHBn-Val adduct; however, the quinone methide is unstable under physiological conditions due to hydrolysis. It was shown that 4-OHBA forms reversible Schiff base adducts with valine, which can be stabilized via reduction in blood generating the 4-OHBn-Val adduct. In addition, trace amounts of isomeric 2-hydroxybenzyl-valine (2-OHBn-Val) adducts were detected in 12 human blood samples (estimated mean adduct level, 5.0 ± 1.4 pmol/g Hb). Further studies are needed to quantify the contributions from identified possible precursor electrophiles to the observed hydroxybenzyl adducts in humans.

The Fate of a Hapten - From the Skin to Modification of Macrophage Migration Inhibitory Factor (MIF) in Lymph Nodes.

Skin (contact) allergy, the most prevalent form of immunotoxicity in humans, is caused by low molecular weight chemicals (haptens) that penetrate stratum corneum and modify endogenous proteins. The fate of haptens after cutaneous absorption, especially what protein(s) they react with, is largely unknown. In this study the fluorescent hapten tetramethylrhodamine isothiocyanate (TRITC) was used to identify hapten-protein conjugates in the local lymph nodes after topical application, as they play a key role in activation of the adaptive immune system. TRITC interacted with dendritic cells but also with T and B cells in the lymph nodes as shown by flow cytometry. Identification of the most abundant TRITC-modified protein in lymph nodes by tandem mass spectrometry revealed TRITC-modification of the N-terminal proline of macrophage migration inhibitory factor (MIF) - an evolutionary well-conserved protein involved in cell-mediated immunity and inflammation. This is the first time a hapten-modified protein has been identified in lymph nodes after topical administration of the hapten. Most haptens are electrophiles and can therefore modify the N-terminal proline of MIF, which has an unusually reactive amino group under physiological conditions; thus, modification of MIF by haptens may have an immunomodulating role in contact allergy as well as in other immunotoxicity reactions.

Evidence for biochemical barrier restoration: Topical solenopsin analogs improve inflammation and acanthosis in the KC-Tie2 mouse model of psoriasis.

Psoriasis is a chronic inflammatory skin disease affecting 2.5-6 million patients in the United States. The cause of psoriasis remains unknown. Previous human and animal studies suggest that patients with a susceptible genetic background and some stimulus, such as barrier disruption, leads to a coordinated signaling events involving cytokines between keratinocytes, endothelial cells, T cells, macrophages and dendritic cells. Ceramides are endogenous skin lipids essential for maintaining skin barrier function and loss of ceramides may underlie inflammatory and premalignant skin. Ceramides act as a double-edged sword, promoting normal skin homeostasis in the native state, but can be metabolized to sphingosine-1-phosphate (S1P), linked to inflammation and tumorigenesis. To overcome this difficulty, we synthesized solenopsin analogs which biochemically act as ceramides, but cannot be metabolized to S1P. We assess their in vivo bioactivity in a well-established mouse model of psoriasis, the KC-Tie2 mouse. Topical solenopsin derivatives normalized cutaneous hyperplasia in this model, decreased T cell infiltration, interleukin (IL)-22 transcription, and reversed the upregulation of calprotectin and Toll-like receptor (TLR) 4 in inflamed skin. Finally, they stimulated interleukin (IL)-12 production in skin dendritic cells. Thus suggesting barrier restoration has both a biochemical and physical component, and both are necessary for optimal barrier restoration.

Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples.

A method we previously developed has been applied to the determination of the glycosylation pattern of specific proteins in biological samples. Six proteins (alpha-1-antitrypsin, transferrin, haptoglobin, C1 inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the expected normal distribution of glycosylation patterns and to assess whether this methodology can be used to discriminate between samples from different individuals. For serum samples, the differences were shown to be small, while much larger differences were found for the CSF samples, with a greater number of glycoforms present. This can be linked to the occurrence of differential glycosylation in proteins expressed in the brain compared with proteins expressed elsewhere in the body. The developed method could distinguish differences in the glycosylation pattern of specific proteins in the individual samples, which was not reflected in the glycan content of total CSF. This is the first time that the glycoforms of several of these proteins have been investigated in CSF.

Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders.

Glycosylation is one of the most common and important post-translational modifications, influencing both the chemical and the biological properties of proteins. Studying the glycosylation of the entire protein population of a sample can be challenging because variations in the concentrations of certain proteins can enhance or obscure changes in glycosylation. Furthermore, alterations in the glycosylation pattern of individual proteins, exhibiting larger variability in disease states, have been suggested as biomarkers for different types of cancer, as well as inflammatory and neurodegenerative diseases. In this paper, we present a rapid and efficient method for glycosylation analysis of individual proteins focusing on changes in the degree of fucosylation or other alterations to the core structure of the glycans, such as the presence of bisecting N-acetylglucosamines and a modified degree of branching. Streptavidin-coated magnetic beads are used in combination with genetically engineered immunoaffinity binders, called VHH antibody fragments. A major advantage of the VHHs is that they are nonglycosylated; thus, enzymatic release of glycans from the targeted protein can be performed directly on the beads. After deglycosylation, the glycans are analyzed by MALDI-TOF-MS. The developed method was evaluated concerning its specificity, and thereafter implemented for studying the glycosylation pattern of two different proteins, alpha-1-antitrypsin and transferrin, in human serum and cerebrospinal fluid. To our knowledge, this is the first example of a protein array-type experiment that employs bead-based immunoaffinity purification in combination with mass spectrometry analysis for fast and efficient glycan analysis of individual proteins in biological fluid.

Peptide Reactivity of Isothiocyanates--Implications for Skin Allergy.

Skin allergy is a chronic condition that affects about 20% of the population of the western world. This disease is caused by small reactive compounds, haptens, able to penetrate into the epidermis and modify endogenous proteins, thereby triggering an immunogenic reaction. Phenyl isothiocyanate (PITC) and ethyl isothiocyanate (EITC) have been suggested to be responsible for allergic skin reactions to chloroprene rubber, the main constituent of wetsuits, orthopedic braces, and many types of sports gear. In the present work we have studied the reactivity of the isothiocyanates PITC, EITC, and tetramethylrhodamine-6-isothiocyanate (6-TRITC) toward peptides under aqueous conditions at physiological pH to gain information about the types of immunogenic complexes these compounds may form in the skin. We found that all three compounds reacted quickly with cysteine moieties. For PITC and 6-TRITC the cysteine adducts decomposed over time, while stable adducts with lysine were formed. These experimental findings were verified by DFT calculations. Our results may suggest that the latter are responsible for allergic reactions to isothiocyanates. The initial adduct formation with cysteine residues may still be of great importance as it prevents hydrolysis and facilitates the transport of isothiocyanates into epidermis where they can form stable immunogenic complexes with lysine-containing proteins.

Honokiol bis-dichloroacetate (Honokiol DCA) demonstrates activity in vemurafenib-resistant melanoma in vivo.

The majority of human melanomas bears BRAF mutations and thus is treated with inhibitors of BRAF, such as vemurafenib. While patients with BRAF mutations often demonstrate an initial dramatic response to vemurafenib, relapse is extremely common. Thus, novel agents are needed for the treatment of these aggressive melanomas. Honokiol is a small molecule compound derived from Magnolia grandiflora that has activity against solid tumors and hematopoietic neoplasms. In order to increase the lipophilicity of honokiol, we have synthesized honokiol DCA, the dichloroacetate ester of honokiol. In addition, we synthesized a novel fluorinated honokiol analog, bis-trifluoromethyl-bis-(4-hydroxy-3-allylphenyl) methane (hexafluoro). Both compounds exhibited activity against A375 melanoma in vivo, but honokiol DCA was more active. Gene arrays comparing treated with vehicle control tumors demonstrated induction of the respiratory enzyme succinate dehydrogenase B (SDHB) by treatment, suggesting that our honokiol analogs induce respiration in vivo. We then examined its effect against a pair of melanomas, LM36 and LM36R, in which LM36R differs from LM36 in that LM36R has acquired vemurafenib resistance. Honokiol DCA demonstrated in vivo activity against LM36R (vemurafenib resistant) but not against parental LM36. Honokiol DCA and hexafluoro inhibited the phosphorylation of DRP1, thus stimulating a phenotype suggestive of respiration through mitochondrial normalization. Honokiol DCA may act in vemurafenib resistant melanomas to increase both respiration and reactive oxygen generation, leading to activity against aggressive melanoma in vivo.

Solenopsin A and analogs exhibit ceramide-like biological activity.

BACKGROUND: (-)-Solenopsin A is a piperidine alkaloid that is a component of the venom of the fire ant Solenopsis invicta. Previously, we have demonstrated that solenopsin exhibit anti-angiogenic activity and downregulate phosphoinositol-3 kinase (PI3K) in the p53 deficient renal cell carcinoma cell line 786-O. Solenopsin has structural similarities to ceramide, a major endogenous regulator of cell signaling and cancer therapy induced apoptosis. METHODS: Different analogs of solenopsin were synthesized in order to explore structure-activity relationships. The anti-proliferative effect of solenopsin and analogs was tested on six different cell lines, including three tumor cell lines, two normal cutaneous cell lines, and one immortalized hyperproliferative cell line. FRET-based reporters were used to study the affect of solenopsin and analogs on Akt activity and PDK1 activation and sucrose density gradient fractionation was performed to examine recruitment of PTEN to membrane rafts. Western-blotting was used to evaluate the affect of solenopsin and analogs on the Akt and the MAPK 44/42 pathways in three different tumor cell lines. Measurement of cellular oxygen consumption rate together with autophagy staining was performed to study mitochondrial function. Finally, the affect of solenopsin and analogs on ROS production was investigated. RESULTS: In this paper we demonstrate that solenopsin analogs with potent anti-proliferative effects can be synthesized from inexpensive dimethylpyridines. To determine whether solenopsin and analogs act as ceramide analogs, we examined the effect of solenopsin and analogs on two stereotypic sites of ceramide activity, namely at lipid rafts and mitochondria. We found that native solenopsin, (-)-solenopsin A, inhibits functional Akt activity and PDK1 activation in lipid rafts in a similar fashion as ceramide. Both cis and trans analogs of solenopsin reduce mitochondrial oxygen consumption, increase reactive oxygen, and kill tumor cells with elevated levels of Akt phosphorylation. However, only solenopsin induces mitophagy, like ceramide. CONCLUSIONS: The requirements for ceramide induced mitophagy and inhibition of Akt activity and PDK1 activation in lipid rafts are under strict stereochemical control. The naturally occurring (-)-solenopsin A mimic some of the functions of ceramide and may be therapeutically useful in the treatment of hyperproliferative and malignant disorders of the skin, even in the presence of elevated levels of Akt.

Ketoprofen-induced formation of amino acid photoadducts: possible explanation for photocontact allergy to ketoprofen.

Photocontact allergy is a well-known side effect of topical preparations of the nonsteroidal anti-inflammatory drug ketoprofen. Photocontact allergy to ketoprofen appears to induce a large number of photocross allergies to both structurally similar and structurally unrelated compounds. Contact and photocontact allergies are explained by structural modification of skin proteins by the allergen. This complex is recognized by the immune system, which initiates an immune response. We have studied ketoprofen's interaction with amino acids to better understand ketoprofen's photoallergenic ability. Irradiation of ketoprofen and amino acid analogues resulted in four different ketoprofen photodecarboxylation products (6-9) together with a fifth photoproduct (5). Dihydroquinazoline 5 was shown to be a reaction product between the indole moiety of 3-methylindole (Trp analogue) and the primary amine benzylamine (Lys analogue). In presence of air, dihydroquinazoline 5 quickly degrades into stable quinazolinone 12. The corresponding quinazolinone (17) was formed upon irradiation of ketoprofen and the amino acids N-acetyl-l-Trp ethyl ester and l-Lys ethyl ester. The formation of these models of an immunogenic complex starts with the ketoprofen-sensitized formation of singlet oxygen, which reacts with the indole moiety of Trp. The formed intermediate subsequently reacts with the primary amino functionality of Lys, or its analogue, to form a Trp-Lys adduct or a mimic thereof. The formation of a specific immunogenic complex that does not contain the allergen but that can still induce photocontact allergy would explain the large number of photocross allergies with ketoprofen. These allergens do not have to be structurally similar as long as they can generate singlet oxygen. To the best of our knowledge, there is no other suggested explanation for ketoprofen's photoallergenic properties that can account for the observed photocross allergies. The formation of a specific immunogenic complex that does not contain the allergen is a novel hypothesis in the field of contact and photocontact allergy.

Investigation of the sunscreen octocrylene's interaction with amino acid analogs in the presence of UV radiation.

Octocrylene is an organic UV filter, commonly used in sunscreens and cosmetics, which can give rise to both contact and photocontact allergy. Our aim was to investigate octocrylene's interaction with amino acid analogs in the presence of UV radiation to better understand the reason for octocrylene's photoallergenic capacity. The amino acid analogs were photolysed in presence and absence of octocrylene for 1 h in cyclohexane. The rate of degradation was considerably slower for all amino acid analogs when octocrylene was present in the mixture. Benzylamine, the lysine analog, did react with octocrylene during the photolysis and the corresponding amide was formed in an acylation reaction. By varying the benzylamine concentration and keeping the octocrylene concentration fixed the reaction rate was shown to be independent of the amine concentration. The same type of acylation reaction took place when octocrylene alone was photolysed in ethanol in which the ethyl ester was formed from octocrylene and ethanol. Our results suggest that octocrylene's ability to cause photocontact allergy could be due to its photoinduced reactivity toward primary amines and alcohols.

Oxidative coupling as a biomimetic approach to the synthesis of scytonemin.

The first total synthesis of the dimeric alkaloid pigment scytonemin is described. The key transformations in its synthesis from 3-indole acetic acid are a Heck carbocyclization and a Suzuki-Miyaura cross-coupling, orchestrated in a stereospecific tandem fashion, followed by a biosynthetically inspired oxidative dimerization. The tandem sequence generates a tetracyclic (E)-3-(arylidene)-3,4-dihydrocyclopenta[b]indol-2(1H)-one that is subsequently dimerized into the unique homodimeric core structure of scytonemin.