title: Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase.
abstract: A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.
authors.affiliation: Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts, U.K.
authors.forename: G M
authors.initials: GM
authors.lastname: Anlezark
authors.forename: R G
authors.initials: RG
authors.lastname: Melton
authors.forename: R F
authors.initials: RF
authors.lastname: Sherwood
authors.forename: W R
authors.initials: WR
authors.lastname: Wilson
authors.forename: W A
authors.initials: WA
authors.lastname: Denny
authors.forename: B D
authors.initials: BD
authors.lastname: Palmer
authors.forename: R J
authors.initials: RJ
authors.lastname: Knox
authors.forename: F
authors.initials: F
authors.lastname: Friedlos
authors.forename: A
authors.initials: A
authors.lastname: Williams
mesh_terms: D000816:Aniline Mustard / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D001388:Aziridines / Q000378:metabolism* / Q000494:pharmacology; D001711:Biotransformation; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009601:Nitroreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011355:Prodrugs / Q000378:metabolism* / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D000970:Antineoplastic Agents; D001388:Aziridines; D011355:Prodrugs; C076205:5-(N,N-bis(2-chloroethyl)amino)-2,4-dinitrobenzamide; C100099:tretazicar; D000816:Aniline Mustard; D009601:Nitroreductases