Gm. Anlezark et al., BIOACTIVATION OF DINITROBENZAMIDE MUSTARDS BY AN ESCHERICHIA-COLI-B NITROREDUCTASE, Biochemical pharmacology, 50(5), 1995, pp. 609-618
A nitroreductase isolated and purified from Escherichia coli B has bee
n demonstrated to have potential applications in ADEPT (antibody-direc
ted enzyme prodrug therapy) by its ability in vitro to reduce dinitrob
enzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bis
chloroethylamino 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 a
dvantage over CB 1954 in that it is not reduced by mammalian DT diapho
rases. Therefore, a series of compounds related to SN 23862 has been s
ynthesized, and evaluated as potential prodrugs both by determination
of kinetic parameters and by ratio of IC50 against UV4 cells when incu
bated in the presence of prodrug, with and without the E. coli enzyme
and cofactor (NADH). Results from the two studies were generally in go
od agreement in that compounds showing no increase in cytotoxicity in
presence of enzyme and cofactor were not substrates for the enzyme. No
ne of the analogues were activated by DT diaphorase isolated from Walk
er 256 carcinoma cells. For those compounds which were substrates for
the E. coil nitroreductase, there was a positive correlation between k
(cat) 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 re
taining similar kinetic characteristics and cytotoxic potency; and SN
25084, where a change in the position of the carboxamide group relativ
e to the mustard resulted in an increased cytotoxicity ratio and k(cat
) compared with SN 23562 (IC50 ratios 214 and 135; k(cat) values of 75
and 26.4 sec(-1), respectively). An analogue (SN 25507) incorporating
both these structural changes had an enhanced k(cat) of 576 sec(-1).
This study elucidates some of the structural requirements of the enzym
e and aids identification of further directions in the search for suit
able prodrugs for an ADEPT nitroreductase system.