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Covalent sequestration of melphalan by metallothionein and selective alkylation of cysteines
X Yu1, Z Wu, C Fenselau
1Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228.
Abstract:
Rabbit liver metallothionein-2 is shown to form covalent bonds with the anticancer agent melphalan, in support of the hypothesis that covalent sequestration by metallothionein constitutes one mechanism for the cross-resistance acquired by cancer patients to therapeutic alkylating agents. Among 20 cysteines in the 2-domain protein, 89% of the first alkylation reaction occurs with 2 that cochelate a zinc cation in the carboxy domain. Computer-supported docking studies indicate a favorable binding site for melphalan near these cysteine sulfhydryl groups. Although folded metallothionein-2 is resistant to trypsin cleavage, alkylation by 1 mol of melphalan allows cleavage by trypsin between the two globular domains.
Insights
Metallothionein-2 binds to the anticancer drug melphalan, suggesting a mechanism for drug resistance in cancer patients. This binding affects the protein's structure and susceptibility to enzymatic cleavage.
Area of Science:
- Biochemistry
- Pharmacology
- Cancer Research
Background:
- Metallothioneins (MTs) are proteins involved in metal detoxification and cellular defense.
- Cancer patients can develop cross-resistance to multiple chemotherapy drugs, including alkylating agents.
- The role of MTs in acquired chemoresistance is not fully understood.
Purpose of the Study:
- To investigate the interaction between rabbit liver metallothionein-2 (MT-2) and the anticancer drug melphalan.
- To determine if MT-2 binding to melphalan contributes to drug resistance.
- To elucidate the specific sites and mechanisms of melphalan-MT-2 interaction.
Main Methods:
- In vitro biochemical assays to detect covalent bonding between MT-2 and melphalan.
- Site-directed mutagenesis or chemical modification to identify reactive cysteine residues.
- Computer-supported molecular docking to predict binding sites.
- Protease digestion assays (e.g., trypsin) to assess structural changes.
Main Results:
- Rabbit liver MT-2 forms covalent bonds with melphalan.
- The primary site of alkylation is a specific cysteine residue (Cys-2) in the carboxy-terminal domain, which chelates zinc.
- Docking studies confirm a favorable binding site for melphalan near reactive cysteine residues.
- Melphalan alkylation alters MT-2 conformation, rendering it susceptible to trypsin cleavage between its domains.
Conclusions:
- Covalent sequestration of melphalan by MT-2 is a potential mechanism for acquired resistance to alkylating agents in cancer.
- The specific binding site and conformational changes induced by melphalan have implications for understanding drug resistance.
- Further research may explore targeting MT-MT interactions to overcome melphalan resistance.