Abstract
Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) used against HIV-1. Currently, NVP is the most widely used anti-HIV drug in developing countries, both in combination therapy and to prevent mother-to-child transmission of HIV. Despite its efficacy against HIV, NVP produces a variety of toxic responses, including hepatotoxicity and skin rash. It is also associated with increased incidences of hepatoneoplasias in rodents. In addition, epidemiological data suggest that NNRTI use is a risk factor for non-AIDS-defining cancers in HIV-positive patients. Current evidence supports the involvement of metabolic activation to reactive electrophiles in NVP toxicity. NVP metabolism includes oxidation to 12-hydroxy-NVP; subsequent Phase II sulfonation produces an electrophilic metabolite, 12-sulfoxy-NVP, capable of reacting with DNA to yield covalent adducts. Since 2’-deoxythymidine (dT) adducts from several alkylating agents are regarded as having significant mutagenic/carcinogenic potential, we investigated the formation of NVP-dT adducts under biomimetic conditions. Toward this goal, we initially prepared and characterized synthetic NVP-dT adduct standards using a palladium-mediated Buchwald-Hartwig coupling strategy. The synthetic standards enabled the identification, by LC-ESI-MS, of 12-(2'-deoxythymidin-N3-yl)-nevirapine (N3-NVP-dT) in the enzymatic hydrolysate of salmon testis DNA reacted with 12-mesyloxy-NVP, a synthetic surrogate for 12-sulfoxy-NVP. N3-NVP-dT, a potentially cytotoxic and mutagenic DNA lesion, was also the only dT-specific adduct detected upon reaction of dT with 12-mesyloxy-NVP. Our data suggest that N3-NVP-dT may be formed in vivo and play a role in the hepatotoxicity and/or putative hepatocarcinogenicity of NVP.
Highlights
The use of non-nucleoside reverse transcriptase inhibitors (NNRTIs) as components of initial combined antiretroviral therapy (CART) is recommended by the World Health Organization guidelines on HIV/AIDS [1]
Resonance and structural assignments were based on the analysis of coupling patterns, including the 13C-1H coupling profiles obtained in bidimensional heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments, performed with standard pulse programs
The coupling reaction of 3',5'-bis-O-(tert-butyldimethylsilyl)-dT with 12-bromo-NVP (8) was performed under previously optimized Buchwald-Hartwig conditions [25], allowing the synthesis and structural characterization of two NVP-derived dT adducts, N3-NVP-dT (9, major) and O4-NVP-dT
Summary
The use of non-nucleoside reverse transcriptase inhibitors (NNRTIs) as components of initial combined antiretroviral therapy (CART) is recommended by the World Health Organization guidelines on HIV/AIDS [1]. While the reasons for the adverse effects of NVP are still unclear, several in vitro and in vivo data are consistent with the involvement of NVP bioactivation, via Phase I oxidation to 12-hydroxy-NVP (2, Figure 1) and subsequent Phase II sulfonation to 12-sulfoxy-NVP (3, Figure 1), in the onset of toxic events elicited by the parent drug [18,19,20,21]. This Phase II metabolite is a reactive electrophile, and expected to react directly with bionucleophiles (e.g., DNA) yielding covalent adducts. To gain insight into the ability of activated NVP derivatives to form dT adducts, we report the synthesis and characterization of NVP-dT adduct standards, and the use of these synthetic standards to identify dT-specific NVP adducts formed in DNA under biologically plausible conditions
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