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A binding site for chlorambucil on metallothionein
1Department of Chemistry and Biochemistry, Structural Biochemistry Center, University of Maryland Baltimore County, 21228, USA.
Biochemistry
|March 5, 1996
Summary
Induced metallothionein (MT) sequesters chlorambucil (CHB) via covalent binding at specific cysteine sites. This interaction, crucial for acquired drug resistance, involves drug modification without altering the protein
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Metallothionein (MT) is implicated in acquired drug resistance.
- The mechanism of MT's role in drug resistance is not fully understood.
- Covalent sequestration is a proposed mechanism for MT's action.
Purpose of the Study:
- To test the hypothesis that induced metallothionein (MT) acts in acquired drug resistance by covalent sequestration.
- To identify the specific sites of covalent modification on MT by chlorambucil (CHB).
- To investigate the impact of drug modification on MT's metal-binding capacity.
Main Methods:
- In vitro incubation of MT with chlorambucil (CHB) to form 1:1 covalent adducts.
- Analysis of proteolytic products of adducts using High-Performance Liquid Chromatography (HPLC) and mass spectrometry.
- Real-time monitoring of the reaction using on-line electrospray ionization with a double-focusing mass spectrometer.
- Molecular docking experiments to predict drug binding selectivity.
Main Results:
- Two major sites of modification on MT were identified: the sulfur atoms of cysteines 33 and 48.
- These modified cysteines co-chelate the same metal atom in native MT.
- Drug-modified MT retains its capacity to bind seven metal ions, similar to unmodified MT.
- Molecular docking revealed that the aziridinium ion in CHB and complementary protein charge densities influence drug binding selectivity.
Conclusions:
- Induced metallothionein (MT) covalently binds chlorambucil (CHB) at specific cysteine residues (Cys33 and Cys48).
- This covalent sequestration mechanism contributes to acquired drug resistance.
- The drug modification does not significantly alter MT's metal-binding stoichiometry.