N-(2-chloroethyl)-N-nitrosourea tethered to lexitropsin induces minor groove lesions at the p53 cDNA that are more cytotoxic than mutagenic

Many different N-chloroethyl-N-nitrosourea (CENU) derivatives have been syn thesized in an attempt to minimize carcinogenic activity while favoring ant ineoplastic activity. CENU derivatives linked to the dipeptide lexitropsin (lex) showed significant changes in groove- and sequence-selective DNA alky lation inducing thermolabile N-3-alkyladenines (N3-Alkyl-As) at lex equilib rium binding sites. CENU-lex sequence specificity for DNA alkylation was de termined using P-32-end-labeled restriction fragments of the p53 cDNA, The adducted sites were converted into single-strand breaks by sequential heati ng at neutral pH and exposure to piperidine, To establish the mutagenic and lethal properties of CENU-lex-specific lesions, a yeast expression vector harboring a human wild-type p53 cDNA was treated in vitro with CENU-lex and transfected into a yeast strain containing the ADE2 gene regulated by a p5 3-responsive promoter. p53 mutants were isolated from independent ade(-) tr ansformants, The results revealed that: (a) CENU-lex preferentially induces N3-Alkyl-A at specific lex equilibrium binding sites, the formations of wh ich are strongly inhibited by distamycin; (b) reactivity toward Gs is still present, albeit to a lesser extent when compared to N-(2-chloroethyl) -N-c yclohexyl-N-nitrosourea and to CENU; (c) 91% of the 49 CENU-lexp53 mutation s (45 of 49) were bp substitutions, 29 of which were GC-->AT transitions, m ainly at 5' purine G sites; (d) all AT-targeted mutations but one were AT-- >TA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GC-->AT transitions were observed, being a real (n = 3, P < 0.0002) CENU-lex mutati on hot spot; and (f) a shift in DNA alkylation sites between lesion spectra induced by CENU-lex and N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea was as sociated with an increased lethality and a decreased mutagenicity, whereas no dramatic change in mutational specificity was observed. Hence, it is tem pting to conclude that, in this experimental system, N3-Alkyl-A is more let hal than mutagenic, Whereas O-6-alkylguanine is a common premutational lesi on formed at non-lex binding sites. These results suggest that CENU derivat ives with virtually absolute specificity for A residues would make targetin g of lethal, nonmutagenic lesions at A+T-rich regions possible, and this ma y represent a new strategy for the development of new chemotherapeutic agen ts with a higher therapeutic index.