Abstract
The research reported here demonstrates covalent binding to the metal-binding protein metallothionein (MT) by the therapeutic nitrogen mustard mechlorethamine. The most surprising aspect of this interaction is the selectivity of the alkylating agent for specific residues of MT. A combination of MS and proteolytic and enzymatic methods was used to deduce specific locations of mechlorethamine alkylation. These experiments indicated that alkylation occurs predominantly in the carboxyl domain of MT, with one molecule of mechlorethamine covalently cross-linking two cysteine residues. Electrospray MS revealed the retention of all seven metal ions in the cross-linked MT/mechlorethamine adducts, highlighting the uniqueness of this protein. Computerized docking experiments supported the hypothesis that selective binding precedes selective alkylation, and the structure of the drug indicates the minimal structural requirements for this binding. These results support the idea that MT overexpressed in tumor cells contributes to the inactivation of anticancer drugs.
Footnotes
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Send reprint requests to: Dr. C. Fenselau, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742.
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This work was supported by the National Institutes of Health (Grants CA67712 and GM21248).
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↵2 The numbering system used here for rabbit liver MT isoform 2a (the 62-amino acid protein) is that used by Fowle and Stillman (1997) and differs from that used in earlier reports from this laboratory, which followed the numbering system of Kagi and Kojima (1987).
- Abbreviations used are::
- MT
- metallothionein
- ACN
- acetonitrile
- CID
- collisionally induced dissociation
- ES
- electrospray ionization
- FAB
- fast atom bombardment
- MALDI
- matrix-assisted laser desorption/ionization
- TFA
- trifluoroacetic acid
- Received March 13, 1998.
- Accepted May 20, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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