Selected Chemistry of Biologically-Active Thiols : N-acetylpenicillamine and 2-mercaptobenzothiazole in Nitrosothiol Formation and Role in allergic contact dermatitis Respectively

Two biologically-active thiols, N-acetylpenicillamine (NAP) and 2-mercaptobenzothiazole (MBT), were studied in this thesis. NAP is known to combine with nitric oxide (NO) to produce S-nitroso-N-acetylpenicillamine (SNAP) and MBT is a known allergen. The formation, reaction dynamics, and detailed kinetics and mechanism of the reaction between nitrous acid (HNO2), prepared in situ, and NAP to produce SNAP were studied. The reaction is first order in nitrite, NAP and acid in pH conditions at or slightly higher than the pKa of HNO2. Both HPLC and quadrupole time-of-flight mass spectrometry techniques confirmed the formation of SNAP and the absence of any other products. Cu(I) ions were found to be effective SNAP-decomposition catalysts. The formation of SNAP occurs through two distinct pathways. One involves the direct reaction of NAP and HNO2 to form SNAP and eliminate water, and the second pathway involved the initial formation of the nitrosyl cation, NO+, which then nitrosates the thiol. The bimolecular rate constant for the reaction of NAP and HNO2 was derived as 2.69 M-1 s-1, while that of direct nitrosation by the nitrosyl cation was 3.00 x 104 M-1 s -1. A simple reaction network made up of four reactions was found to be sufficient in simulating the formation kinetics and acid-induced decomposition of SNAP. The chemical mechanism leading to MBT\u27s allergenicity is unknown. It was hypothesized that the thiol group is critical to MBT\u27s covalent binding/haptenation to nucleophilic protein residues. Hypochlorous acid oxidized MBT to the disulfide, 2, 2\u27-dithiobis(benzothiazole) (MBTS), within the glove matrix. Cysteine reduced MBTS to MBT with subsequent formation of the mixed disulfide 2-amino-3-(benzothiazol-2-yl disulfanyl)-propionic acid. Simultaneous reduction of MBTS and disulfide formation with Cys34 on bovine serum albumin was observed, suggesting a potential route of protein haptenation through covalent bonding between cysteinyl residues on proteins and the MBT/MBTS thiol moiety. Guinea pigs were sensitized to MBT using a modified guinea pig maximization assay (GPMT) and cross-reactivity towards MBTS and the free thiol-lacking or blocked compounds benzothiazole (BT), 2-hydroxybenzothiazole (HBT) and 2-(methylthio)benzothiazole (MTBT) assessed. MBT and MBTS, but not BT, HBT or MTBT elicited allergic contact dermatitis (ACD) in MBT-sensitized animals

A New Pathway for Protein Haptenation by beta-Lactams

"This is the peer reviewed version of the following article: Pérez-Ruíz, Raúl, Emilio Lence, Inmaculada Andreu, Daniel Limones-Herrero, Concepción González-Bello, Miguel A. Miranda, and M. Consuelo Jiménez. 2017. A New Pathway for Protein Haptenation by β-Lactams. Chemistry - A European Journal 23 (56). Wiley: 13986 94. doi:10.1002/chem.201702643, which has been published in final form at https://doi.org/10.1002/chem.201702643. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."[EN] The covalent binding of beta-lactams to proteins upon photochemical activation has been demonstrated by using an integrated approach that combines photochemical, proteomic and computational studies, selecting human serum albumin (HSA) as a target protein and ezetimibe (1) as a probe. The results have revealed a novel protein haptenation pathway for this family of drugs that is an alternative to the known nucleophilic ring opening of beta-lactams by the free amino group of lysine residues. Thus, photochemical ring splitting of the beta-lactam ring, following a formal retro-Staudinger reaction, gives a highly reactive ketene intermediate that is trapped by the neighbouring lysine residues, leading to an amide adduct. For the investigated 1/HSA system, covalent modification of residues Lys414 and Lys525, which are located in sub-domains IIIA and IIIB, respectively, occurs. The observed photobinding may constitute the key step in the sequence of events leading to photoallergy. Docking and molecular dynamics simulation studies provide an insight into the molecular basis of the selectivity of 1 for these HSA sub-domains and the covalent modification mechanism. Computational studies also reveal positive cooperative binding of sub-domain IIIB that explains the experimentally observed modification of Lys414, which is located in a barely accessible pocket (sub-domain IIIA).Financial support from Ministerio de Economia, Industria y Competitividad (CTQ2013-47872-C2-1-P, CTQ2016-78875-P, SAF2013-42899-R, SAF2016-75638-R), Instituto de Salud Carlos III (RD12/0013/0009 and RD16/0006/0030), Generalitat Valenciana (PROMETEOII/2013/005), Xunta de Galicia (Centro singular de investigacion de Galicia accreditation 2016-2019, ED431G/09) and European Union (European Regional Development Fund -ERDF) is gratefully acknowledged. E.L. thanks the Xunta de Galicia for a postdoctoral fellowship. We are grateful to the Centro de Supercomputacion de Galicia (CESGA) for use of the Finis Terrae II supercomputer. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed PRB2-ISCIII and is supported by grant PT13/0001, of the PE I+D+i 2013-2016, funded by ISCIII and FEDER.Pérez-Ruiz, R.; Lence, E.; Andreu Ros, MI.; Limones Herrero, D.; González-Bello, C.; Miranda Alonso, MÁ.; Jiménez Molero, MC. (2017). A New Pathway for Protein Haptenation by beta-Lactams. Chemistry - A European Journal. 23(56):13986-13994. https://doi.org/10.1002/chem.201702643S13986139942356Van Boeckel, T. P., Gandra, S., Ashok, A., Caudron, Q., Grenfell, B. T., Levin, S. A., & Laxminarayan, R. (2014). Global antibiotic consumption 2000 to 2010: an analysis of national pharmaceutical sales data. The Lancet Infectious Diseases, 14(8), 742-750. doi:10.1016/s1473-3099(14)70780-7Elander, R. P. (2003). Industrial production of β-lactam antibiotics. 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Developing a framework for assessing respiratory sensitization: A workshop report

Respiratory tract sensitization can have significant acute and chronic health implications. While induction of respiratory sensitization is widely recognized for some chemicals, validated standard methods or frameworks for identifying and characterizing the hazard are not available. A workshop on assessment of respiratory sensitization was held to discuss the current state of science for identification and characterization of respiratory sensitizer hazard, identify information facilitating development of validated standard methods and frameworks, and consider the regulatory and practical risk management needs. Participants agreed on a predominant Th2 immunological mechanism and several steps in respiratory sensitization. Some overlapping cellular events in respiratory and skin sensitization are well understood, but full mechanism(s) remain unavailable. Progress on non-animal approaches to skin sensitization testing, ranging from in vitro systems, –omics, in silico profiling, and structural profiling were acknowledged. Addressing both induction and elicitation phases remains challenging. Participants identified lack of a unifying dose metric as increasing the difficulty of interpreting dosimetry across exposures. A number of research needs were identified, including an agreed list of respiratory sensitizers and other asthmagens, distinguishing between adverse effects from immune-mediated versus non immunological mechanisms. A number of themes emerged from the discussion regarding future testing strategies, particularly the need for a tiered framework respiratory sensitizer assessment. These workshop present a basis for moving towards a weight-of-evidence assessment

Reactivity Measurement in Estimation of Benzoquinone and Benzoquinone Derivatives’ Allergenicity

Benzoquinone (BQ) and benzoquinone derivatives (BQD) are used in the production of dyes and cosmetics. While BQ, an extreme skin sensitizer, is an electrophile known to covalently modify proteins via Michael Addition (MA) reaction whilst halogen substituted BQD undergo nucleophilic vinylic substitution (SNV) mechanism onto amine and thiol moieties on proteins, the allergenic effects of adding substituents on BQ have not been reported. The effects of inserting substituents on the BQ ring has not been studied in animal assays. However, mandated reduction/elimination of animals used in cosmetics testing in Europe has led to an increased need for alternatives for the prediction of skin sensitization potential. Electron withdrawing and electron donating substituents on BQ were assessed for effects on BQ reactivity toward nitrobenzene thiol (NBT). The NBT binding studies demonstrated that addition of EWG to BQ as exemplified by the chlorine substituted BQDs increased reactivity while addition of EDG as in the methyl substituted BQDs reduced reactivity. BQ and BQD skin allerginicity was evaluated in the murine local lymph node assay (LLNA). BQD with electron withdrawing groups had the highest chemical potency followed by unsubstituted BQ and the least potent were the BQD with electron donating groups. The BQD results demonstrate the impact of inductive effects on both BQ reactivity and allergenicity, and suggest the potential utility of chemical reactivity data for electrophilic allergen identification and potency ranking

Characterization and Comparative Analysis of 2,4-Toluene Diisocyanate and 1,6-Hexamethylene Diisocyanate Haptenated Human Serum Albumin and Hemoglobin

Diisocyanates (dNCOs) are lowmolecularweight chemical sensitizers that reactwith autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCOspecific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may bemodified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb–TDI antigenicity was approximately 30% that of HSA–TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity