Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase

Area of Science:

• Biochemistry
• Enzyme kinetics
• Drug discovery

Background:

• Antibody-directed enzyme prodrug therapy (ADEPT) utilizes enzymes to activate prodrugs selectively at tumor sites.
• Escherichia coli nitroreductase shows promise for ADEPT by reducing dinitrobenzamides to cytotoxic agents.
• Mammalian DT-diaphorases can inactivate some prodrugs, necessitating the development of enzyme-specific substrates.

Purpose of the Study:

• To synthesize and evaluate novel dinitrobenzamide analogues as prodrugs for an E. coli nitroreductase-based ADEPT system.
• To assess the kinetic parameters and cytotoxicity of these prodrugs in the presence and absence of the E. coli nitroreductase.
• To identify prodrugs with improved characteristics, such as enhanced solubility and selective activation, compared to existing compounds like CB 1954 and SN 23862.

Main Methods:

• Purification and characterization of nitroreductase from Escherichia coli B.
• Synthesis of a series of dinitrobenzamide analogues based on SN 23862.
• In vitro enzymatic assays to determine kinetic parameters (kcat) and prodrug activation by E. coli nitroreductase and mammalian DT-diaphorase.
• Cytotoxicity assays (IC50) using UV4 cells incubated with prodrugs, with and without the enzyme and cofactor (NADH).

Main Results:

• SN 23862 is selectively reduced by E. coli nitroreductase to a hydroxylamine that forms DNA interstrand crosslinks.
• None of the synthesized analogues were activated by mammalian DT-diaphorase, indicating potential for selective tumor targeting.
• Compounds SN 25261 and SN 25084 demonstrated advantageous properties, with SN 25261 showing increased aqueous solubility and SN 25084 exhibiting a higher cytotoxicity ratio and kcat.
• Analogue SN 25507, combining structural features of SN 25261 and SN 25084, exhibited a significantly enhanced kcat.

Conclusions:

• The study identified key structural requirements for prodrugs activated by E. coli nitroreductase for ADEPT.
• Novel prodrug candidates, particularly SN 25261 and SN 25084, show promise for improved ADEPT efficacy due to enhanced solubility, selective activation, and potent cytotoxicity.
• Further development of nitroreductase-activated prodrugs holds significant potential for targeted cancer therapy.