Toshikazu Bando

Molecular design of a pyrrole-imidazole hairpin polyamides for effective DNA alkylation.

New hairpin polyamide-CPI (CPI = cyclopropylpyrroloindole) conjugates, compounds 12-14, were synthesized and their DNA-alkylating activities compared with the previously prepared hairpin polyamide, compound 1, by high-resolution denaturing gel electrophoresis with 450 base pair (bp) DNA fragments and by HPLC product analysis of the synthetic decanucleotide. In accord with our previous results, alkylation by compound 1 occurred predominantly at the G moiety of the sequence 5-AGTCAG-3 (site 3). However, compound 12, in which the structure of the alkylating moiety of compound 1 is replaced with segment A of duocarmycin A DU-86 (CPI), did not show any DNA alkylating activity. In clear contrast, the hairpin CPI conjugate 13, which differs from compound 1 in that it lacks one Py unit and possesses a vinyl linker, alkylated the A of 5-AGTCAG-3 (site 3) efficiently at nanomolar concentrations. Alkylation by compound 14, which has a vinyl linker, occurred at the A of 5-AGTCCA-3 (site 6) and at several minor alkylation sites, including mismatch alkylation at A of 5-TCACAA-3 (site 2). The significantly different reactivity of the alkylating hairpin polyamides 1, 12, 13, and 14 was further confirmed by HPLC product analysis by using a synthetic decanucleotide. The results suggest that hairpin polyamide--CPI conjugate 13 alkylates effectively according to Dervans pairing rule, and with a new mode of recognition in which the Im-vinyl linker (L) pair targets G-C base pairs. These results demonstrate that incorporation of the vinyl-linker pairing with Im dramatically improves the reactivity of hairpin polyamide--CPI conjugates.

Sequence specificity, reactivity, and antitumor activity of DNA-alkylating pyrrole-imidazole diamides.

Three conjugates of imidazole (Im)-pyrrole (Py) diamide and a DNA-alkylating moiety derived from the antibiotic duocarmycin A were synthesized, and their sequence specificity, reactivity, and antitumor activity comparatively examined. Sequencing gel analysis indicated that ImPyDu (1) alkylates DNA at the 3 end of AT-rich sequences at micromolar concentration. ImPyDu86 (2) reacts with DNA at AT-rich sites together with dialkylation sites at micromolar concentration. ImPyLDu86 (3) efficiently alkylates dialkylation sites at nanomolar concentration. Average values of log IC(50) against a 39 cancer cell line panel of 1-3 were -4.59, -5.95, and -8.25, respectively. The differential growth inhibition pattern of 1-3 varied with relatively low correlation coefficients. Array-based gene expression monitoring was performed for 3 in a human lung cancer cell line. Substantial downregulation of expression was seen for genes involved in DNA damage response, transcription, and signal transduction.

Specific alkylation of human telomere repeats by hairpin pyrrole-imidazole polyamide.

A novel hairpin polyamide-cyclopropapyrroloindole (CPI) conjugate PyImImIm-gamma-PyPyPyLDu86 (conjugate 11), which targets human telomere repeats d(TTAGGG)(n)/d(CCCTAA)(n), was synthesized. High resolution denaturing polyacrylamide gel electrophoresis using 44 bp DNA fragments and HPLC product analysis of a synthetic nonanucleotide demonstrated that conjugate 11 alkylates the target adenine in the telomere repeats, 5-CCCTAA-3. Examination of the antitumor activity of conjugate 11 using a panel of 39 cancer cell lines demonstrated that the average concentration of conjugate 11 required for 50% growth inhibition was 5.75 microM, which is superior to pepleomycin and bleomycin and comparable to cisplatin.

Cover Picture: Chem. Eur. J. 20/2002

The cover picture shows the energy-minimized structure of the dCAAGTCAGAG/dCTCTGACTTG pyrrole–imidazole (blue and red, respectively, hairpin CPI conjugate complex (CPI = cyclopropylpyrroloindole). Minimization was performed in the presence of 18 sodium cations and a 10 A layer of H2O by CFF force-field parameters. Each strand of DNA is drawn in white and ribbon representation is drawn in light blue. The CPI conjugate effectively alkylated the A moiety (purple) of 5′-AGTCA-3′ site at nanomolar concentration. Further work is described in more detail by H. Sugiyama et al. on p. 4781u2009ff.

Molecular design of hairpin imidazole-pyrrole polyamide for effective DNA alkylation

A new type of hairpin polyamide-CPI conjugate possessing a vinyl linker has been synthesized. Sequence-selective alkylation of double-stranded DNA by conjugates was investigated by high resolution denaturing gel electrophoresis using 450 bp DNA fragments. Highly-efficient DNA alkylation predominantly occurs at the purines of 5-(A/T)G(T/A)CPu-3 site at nanomolar concentration.