Avaliação da toxicidade do fármaco anti-HIV abacavir: síntese e caracterização estrutural de possíveis biomarcadores

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em BioorgânicaA oxidação de Swern do abacavir permitiu a preparação dos dois aldeídos que se pensam estar envolvidos na via metabólica deste fármaco anti-HIV, o (S)-4-[2`-amino-6`- (ciclopropilamino)-9H-purin-9`-il]ciclopent-1-enilcarbaldeído (aldeído conjugado) e o (S)-4-[2`-amino-6`-(ciclopropilamino)-9H-purin-9`-il]ciclopent-2-enilcarbaldeído (aldeído não conjugado). A reactividade do aldeído conjugado foi estudada in vitro na presença de vários bionucleófilos. Na reacção com a lisina, após tratamento com cianoboro-hidreto de sódio, foi possível detectar a formação de um aducto covalente, por LC-MS. No entanto, apesar das diversas tentativas para isolar este aducto por HPLC semi-preparativo, não foi possível isolálo em quantidade suficiente para possibilitar a sua caracterização por RMN. Similarmente, na reacção de modificação do valinato de etilo com este derivado electrófilo, seguida de redução, o aducto formado foi apenas detectado por LC-MS. No entanto, com o tratamento desta mistura reaccional com fenilisotiocianato (degradação de Edman) foi possível simplificar a mistura reaccional possibilitando o isolamento dum aducto sob a forma de uma feniltiohidantoína, com um rendimento de 15%, que foi caracterizado estruturalmente por RMN e EM. Curiosamente, quando se modificou o valinato de etilo com o aldeído não conjugado obteve-se a mesma tio-hidantoína. Este resultado sugere que o aldeído não conjugado quando formado in vivo rapidamente isomeriza ao aldeído conjugado, termodinamicamente mais estável. Assim, será o aldeído conjugado o intermediário electrófilo responsável pela reacção com os bionucleófilo que leva à formação de aductos covalentes, via base de Schiff, que se pensam estar na origem das reacções tóxicas associadas ao abacavir. A feniltioidantoína preparada foi posteriormente utilizada como padrão para monitorizar, por LC-MS, a formação de aductos com os resíduos de valina N-terminal da Hemoglobina (Hb), formados por modificação desta proteína com o aldeído conjugado seguida de degradação de Edman. A incubação do abacavir na fracção citosólica de fígado de rato, seguida de adição de valinato de etilo, redução e degradação de Edman permitiu a identificação por LC-MS, por comparação com o padrão, da mesma feniltioidantoína. Estes resultados preliminares sugerem que a via metabólica do abacavir que conduz à formação do 1’’-carboxilato, via um intermediário aldeído, poderá estar ligada às reacções de hipersensibilidade associadas ao abacavir. Adicionalmente, o aducto formado com a valina Nterminal da Hb, sendo de fácil monitorização, poderá ser um possível biomarcador de toxicidade do abacavir

A Metabolomics-Inspired Strategy for the Identification of Protein Covalent Modifications

This work was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal, through projects UID/QUI/00100/2019, IF/01091/2013/CP1163/CT0001 and PTDC/QUIQAN/32242/2017 as well as doctoral fellowships SFRH/BD/102846/2014 (to CC) and SFRH/BD/140157/2018 (to JN);joint funding from FCT and the COMPETE Program is also acknowledge through RNEM-LISBOA-01-0145-FEDER-022125-funded postdoctoral fellowship (to JM).Identification of protein covalent modifications (adducts) is a challenging task mainly due to the lack of data processing approaches for adductomics studies. Despite the huge technological advances in mass spectrometry (MS) instrumentation and bioinformatics tools for proteomics studies, these methodologies have very limited success on the identification of low abundant protein adducts. Herein we report a novel strategy inspired on the metabolomics workflows for the identification of covalently-modified peptides that consists on LC-MS data preprocessing followed by statistical analysis. The usefulness of this strategy was evaluated using experimental LC-MS data of histones isolated from HepG2 and THLE2 cells exposed to the chemical carcinogen glycidamide. LC-MS data was preprocessed using the open-source software MZmine and potential adducts were selected based on the m/z increments corresponding to glycidamide incorporation. Then, statistical analysis was applied to reveal the potential adducts as those ions are differently present in cells exposed and not exposed to glycidamide. The results were compared with the ones obtained upon the standard proteomics methodology, which relies on producing comprehensive MS/MS data by data dependent acquisition and analysis with proteomics data search engines. Our novel strategy was able to differentiate HepG2 and THLE2 and to identify adducts that were not detected by the standard methodology of adductomics. Thus, this metabolomics driven approach in adductomics will not only open new opportunities for the identification of protein epigenetic modifications, but also adducts formed by endogenous and exogenous exposure to chemical agents.publishersversionpublishe

Mass Spectrometry-Based Methodologies for Targeted and Untargeted Identification of Protein Covalent Adducts (Adductomics): Current Status and Challenges

Protein covalent adducts formed upon exposure to reactive (mainly electrophilic) chemicals may lead to the development of a wide range of deleterious health outcomes. Therefore, the identification of protein covalent adducts constitutes a huge opportunity for a better understanding of events underlying diseases and for the development of biomarkers which may constitute effective tools for disease diagnosis/prognosis, for the application of personalized medicine approaches and for accurately assessing human exposure to chemical toxicants. The currently available mass spectrometry (MS)-based methodologies, are clearly the most suitable for the analysis of protein covalent modifications, providing accuracy, sensitivity, unbiased identification of the modified residue and conjugates along with quantitative information. However, despite the huge technological advances in MS instrumentation and bioinformatics tools, the identification of low abundant protein covalent adducts is still challenging. This review is aimed at summarizing the MS-based methodologies currently used for the identification of protein covalent adducts and the strategies developed to overcome the analytical challenges, involving not only sample pre-treatment procedures but also distinct MS and data analysis approaches

Covalent histone modification by an electrophilic derivative of the anti-HIV drug nevirapine

Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor widely used in combined antiretroviral therapy and to prevent mother-to-child transmission of the human immunodeficiency virus type 1, is associated with several adverse side effects. Using 12-mesyloxy-nevirapine, a model electrophile of the reactive metabolites derived from the NVP Phase I metabolite, 12-hydroxy-NVP, we demonstrate that the nucleophilic core and C-terminal residues of histones are targets for covalent adduct formation. We identified multiple NVP-modification sites at lysine (e.g., H2BK47, H4K32), histidine (e.g., H2BH110, H4H76), and serine (e.g., H2BS33) residues of the four histones using a mass spectrometry-based bottom-up proteomic analysis. In particular, H2BK47, H2BH110, H2AH83, and H4H76 were found to be potential hot spots for NVP incorporation. Notably, a remarkable selectivity to the imidazole ring of histidine was observed, with modification by NVP detected in three out of the 11 histidine residues of histones. This suggests that NVP-modified histidine residues of histones are prospective markers of the drug's bioactivation and/or toxicity. Importantly, NVP-derived modifications were identified at sites known to determine chromatin structure (e.g., H4H76) or that can undergo multiple types of post-translational modifications (e.g., H2BK47, H4H76). These results open new insights into the molecular mechanisms of drug-induced adverse reactions.This work was supported in part by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme. We also thank Fundação para a Ciência e a Tecnologia (FCT), Portugal, for financial support through projects UID/QUI/00100/2020 (to CQE), RECI/QEQ-MED/0330/2012 and PTDC/QUI-QAN/32242/2017, as well as contract CEECIND/02001/2017 (to A.M.M.A) and doctoral fellowships SFRH/BD/80690/2011 (to SGH), SFRH/BD/75426/2010 (to ILM), and SFRH/BD/102846/2014 (to CC). Joint funding from FCT and the COMPETE Program through grant SAICTPAC/0019/2015 and RNEM-LISBOA-01-0145-FEDER-022125 funding are also gratefully acknowledged. The CRG/UPF Proteomics Unit is part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)” supported by grant PT13/0001 of the Instituto de Salud Carlos III (ISCIII

Bis-alkylamine Indolo[3,2‑<i>b</i>]quinolines as Hemozoin Ligands: Implications for Antimalarial Cytostatic and Cytocidal Activities

To get insight into the relevance of targeting hemozoin (Hz) crystals, two isomeric series, N5,N10-bis-alkylamine (<b>2a</b>–<b>k</b>) and N10,O11-bis-alkylamine (<b>3a</b>–<b>k</b>) indolo­[3,2-<i>b</i>]­quinolines, were evaluated for their in vitro activity against chloroquine (CQ)-resistant and sensitive strains of <i>Plasmodium falciparum</i>. In general, compounds of series <b>3</b> were more active than isomers <b>2</b>, with IC₅₀/LD₅₀ ranging from 25/233 nM (<b>3i</b>) to 1.3 (<b>3a</b>)/10.7 (<b>3b</b>) μM. SAR analyses showed that lipophilicity and chlorine substitution at C3 increased both cytostatic and cytocidal activities. Both series bound to hematin monomer, inhibited β-hematin formation in vitro, delayed intraerythrocytic parasite development with apparent inhibition of Hz biocrystallization, and showed higher cytocidal activity against schizonts. In addition, cytostatic and cytocidal activities of series <b>3</b>, but not those of isomers <b>2</b>, correlated with calculated vacuole accumulation ratios, suggesting different capacities of <b>2</b> and <b>3</b> to bind to the Hz crystal face {001} exposed on the vacuole aqueous medium and different mechanisms of cytocidal potency

Selenium-Containing Chrysin and Quercetin Derivatives: Attractive Scaffolds for Cancer Therapy

Selenium-containing chrysin (<b>SeChry</b>) and 3,7,3′,4′-tetramethylquercetin (<b>SePQue</b>) derivatives were synthesized by a microwave-based methodology. In addition to their improvement in terms of DPPH scavenging and potential GPx-like activities, when tested in a panel of cancer cell lines both selenium-derivatives revealed consistently to be more cytotoxic when compared with their oxo and thio-analogues, evidencing the key role of selenocabonyl moiety for these activities. In particular, SeChry elicited a noteworthy cytotoxic activity with mean IC₅₀ values 18- and 3-fold lower than those observed for chrysin and cisplatin, respectively. Additionally, these seleno-derivatives evidenced an ability to overcome cisplatin and multidrug resistance. Notably, a differential behavior toward malignant and nonmalignant cells was observed for <b>SeChry</b> and <b>SePQue</b>, exhibiting higher selectivity indexes when compared with the chalcogen-derivatives and cisplatin. Our preliminary investigation on the mechanism of cytotoxicity of <b>SeChry</b> and <b>SePQue</b> in MCF-7 human mammary cancer cells demonstrated their capacity to efficiently suppress the clonal expansion along with their ability to hamper TrxR activity leading to apoptotic cell death