Protein Targets of Reactive Electrophiles in Human Liver Microsomes

The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan, is commonly attributed to their ability to induce DNA-DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA-protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA-protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here we show that the DNA repair protein, O 6 -alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32 P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with guanine half mustards of chlorambucil or mechlorethamine revealed the ability of the protein to form either one or two cross-links to guanine. C145A AGT -a variant containing a single point mutation in the protein's active site -was found capable of forming a single guanine conjugate, while cross-linking was virtually abolished upon treatment of the C145A/C150S AGT double mutant with the guanine half mustards. HPLC-ESI + -MS/MS sequencing of the tryptic peptides obtained from the wild type AGT protein that had been treated with nitrogen mustards in the presence of DNA confirmed that the cross-linking took place between the N7 position of guanine in DNA and two active site residues within the AGT protein (Cys 145 and Cys 150 ). The exact chemical structures of AGT-DNA cross-links induced by chlorambucil and mechlorethamine were identified as N-(2-[Scysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)-p-aminophenylbuyric acid and N-(2-[Scysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)methylamine, respectively, based upon HPLC-MS/MS analysis of protein hydrolysates in parallel with the corresponding amino acid conjugates prepared synthetically. Mechlorethamine-induced AGT-DNA conjugates were isolated from protein extracts of AGT-*To whom correspondence should be addressed: University of Minnesota Cancer Center, 420 Delaware St SE -MMC 806, Minneapolis, MN 55455, USA. ph: 612-626-3432 fax: 612-626-5135 trety001@umn.edu. Supporting Information Available: Synthetic procedures for the preparation of the guanine half mustards and amino acid-guanine conjugates of chlorambucil and mechlorethamine, SDS-PAGE analysis of nitrogen mustard-induced histone H4-DNA cross-links, MS spectra of histone H4 following exposure to guanine half mustards, MS/MS spectra of AGT tryptic peptides containing nitrogen mustardinduced lesions, and amino acid sequences of human AGT variants and bovine histone H4 can be found in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs...

Section: Detection Of Nitrogen Mustard-induced Agt-dna Cross-links Inmentioning

confidence: 99%

Cross-Linking of the DNA Repair Protein O⁶-Alkylguanine DNA Alkyltransferase to DNA in the Presence of Antitumor Nitrogen Mustards

Loeber

Michaelson

Fang

et al. 2008

“…Research on mechanistic aspects of cellular damage caused by chemically reactive drug metabolites holds the potential of dramatically altering the way that the pharmaceutical industry currently deals with metabolic activation issues, although studies in this area face formidable challenges in deciphering the critical molecular targets for injury and their role in signaling pathways for cellular defense or cell death. Recent publications on the application of MS-based proteomics techniques to the problem of reactive metabolite-protein covalent adducts underscore the complexity of the issue but also suggest that the tools are now available to identify key cellular targets of these electrophiles, a knowledge of that could lead to future predictive approaches to toxicities mediated by reactive intermediates (46,(70)(71)(72)(73). Of particular importance in this area is the recognition that different reactive electrophiles can exhibit a relatively high selectivity for alkylation of different proteins, and for different nucleophilic sites within these proteins, the toxicological significance of which remains to be established.…”

Section: Drug Metabolism In the Future: What Lies Ahead?mentioning

confidence: 99%

Metabolism and Toxicity of Drugs. Two Decades of Progress in Industrial Drug Metabolism

Baillie

2007

183

116

The science of drug metabolism and pharmacokinetics (DMPK) has developed significantly over the past 20 years, and its functional role in today's pharmaceutical industry has matured to the point where DMPK has become an indispensable discipline in support of drug discovery and development. While contributions to the lead optimization phase of discovery efforts have been particularly noteworthy in helping to select only the best drug candidates for entry into development, it should be recognized that the scope of DMPK spans the continuum of discovery through clinical evaluation and even into the post-marketing phase; as such, the breadth of DMPK's involvement is almost unique in contemporary pharmaceutical research. This perspective summarizes notable advances in the field, many of which have been made possible by technological developments in areas such as molecular biology, genetics, and bioanalytical chemistry, and highlights the critical nature of key partnerships between Drug Metabolism, Medicinal Chemistry, and Safety Assessment groups in attempting to advance drug candidates with a low potential for causing adverse events in humans. Finally, some speculative predictions are made of the future role of DMPK in pharmaceutical research, where current advances in our mechanistic understanding of the molecular processes that control the absorption, disposition, metabolism, elimination, and toxicity of drugs and their biotransformation products will combine to further enhance the impact of DMPK in drug discovery and development.

“…We have recently used biotinylated electrophile probes to investigate the scope and characteristics of protein covalent binding to subcellular proteomes (30,31). The iodoacetamido probe N-iodoacetyl-N-biotinylhexylenediamine (IAB) and Nalkylmaleimido probe 1-biotinamido-4-(4′-[maleimidoethylcyclo-hexane]carboxamido)butane (BMCC) (Figure 1) display reaction chemistries analogous to many electrophilic metabolites and endogenous electrophiles.…”

Section: Introductionmentioning

confidence: 99%

“…The iodoacetamido probe N-iodoacetyl-N-biotinylhexylenediamine (IAB) and Nalkylmaleimido probe 1-biotinamido-4-(4′-[maleimidoethylcyclo-hexane]carboxamido)butane (BMCC) (Figure 1) display reaction chemistries analogous to many electrophilic metabolites and endogenous electrophiles. Adducts formed with these probes have been mapped to over a thousand specific cysteines in cytoplasmic, nuclear, and microsomal proteins (30,31). Moreover, adduction appears to be associated with different biological effects, as IAB, but not BMCC-induced endoplasmic reticulum (ER) stress in cells exposed to these probes (31).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Mitochondrial Protein Targets of Thiol-Reactive Electrophiles

Wong

Liebler

2008

Self Cite

Mitochondria serve a pivotal role in the regulation of apoptosis or programmed cell death. Recent studies have demonstrated that reactive electrophiles induce mitochondrion-dependent apoptosis. We hypothesize that covalent modification of specific mitochondrial proteins by reactive electrophiles serves as a trigger leading to the initiation of apoptosis. In this study, we identified protein targets of the model biotin-tagged electrophile probes N-iodoacetyl-N-biotinylhexylene-diamine (IAB) and 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]carboxamido)butane (BMCC) in HEK293 cell mitochondrial fractions by liquid chromatography-tandem mass spectrometry (LC-MS-MS). These electrophiles reproducibly adducted a total of 1693 cysteine residues that mapped to 809 proteins. Protein modifications were selective in that only 438 cysteine sites in 1255 cysteinyl peptide adducts (35%) and 362 of the 809 identified protein targets (45%) were adducted by both electrophiles. Of these, approximately one-third were annotated to the mitochondria following protein database analysis. IAB initiated apoptotic events including cytochrome c release, caspase-3 activation, and poly(ADP-ribose)polymerase (PARP) cleavage, whereas BMCC did not. Of the identified targets of IAB and BMCC, 44 were apoptosis-related proteins, and adduction site specificity on these targets differed between the two probes. Differences in sites of modification between these two electrophiles may reveal alkylation sites whose modification triggers apoptosis.