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We designed and synthesized human telomere alkylating N-methylpyrrole-N-methylimidazole (PI) polyamide conjugates (1-6). The C-type conjugates 1-3 possessed a chlorambucil moiety at the C terminus, whereas the N-type conjugates 4-6 had one of these moieties at the N terminus. The DNA alkylating activity of these conjugates was evaluated by high-resolution denaturing polyacrylamide gel electrophoresis using a 220bp DNA fragment containing the human telomere repeat sequence 5'-(GGGTTA)(4)-3'/5'-(TAACCC)(4)-3'. C-type conjugates are designed to alkylate the G-rich-strand-containing 5'-GGGTTA-3' and N-type conjugates were designed to alkylate the complementary C-rich strand-containing 5'-TAACCC-3' sequence. The difference between conjugates 1-3 and 4-6 lies in the linker region between the polyamide moiety and chlorambucil. Conjugates 1 and 4 efficiently alkylated the 5'-GGTTAGGGTTA-3' and 5'-CCCTAACCCTAA-3' sequences, respectively, by recognizing 11bp in the presence of distamycin A (Dist), in a heterotrimeric manner: one long alkylating polyamide conjugate (1-6) and two short partners (Dist)., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In recent years, many diseases including cancer and hereditary and viral diseases have been understood at the DNA sequence level. Direct control of the expression level of a specific gene would provide a promising approach for knowledge-based therapy. N-methylpyrrole and N-methylimidazole polyamides are a new type of small compound that precisely bind to the minor groove of the DNA duplex in a sequence-specific fashion and recruit alkylating agents to the target sequence. We designed and synthesized a series of sequence-specific alkylating Py-Im polyamide conjugates that selectively alkylate predetermined DNA sequences. We have shown that sequence-specific alkylating agents possess gene-silencing activities when they alkylate coding regions of template strands and show promising potency against human cancer cell lines and xenografts possessing human cancer cells. In this study, we focus on recent progress in alkylating Py-Im polyamides with regard to sequence specificity and biological activities, and the future direction of the rational molecular design of genetic switches in the post-genome era is described.

We investigated sequence-specific DNA alkylation using conjugates between the N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide and the DNA alkylating agent, chlorambucil, or 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). Polyamide-chlorambucil conjugates 1-4 differed in the position at which the DNA alkylating chlorambucil moiety was bound to the Py-Im polyamide. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) revealed that chlorambucil conjugates 1-4 alkylated DNA at the sequences recognized by the Py-Im polyamide core moiety. Reactivity and sequence specificity were greatly affected by the conjugation position, which reflects the geometry of the alkylating agent in the DNA minor groove. Polyamide-seco-CBI conjugate 5 was synthesized to compare the efficacy of chlorambucil with that of seco-CBI as an alkylating moiety for Py-Im polyamides. Denaturing PAGE analysis revealed that DNA alkylation activity of polyamide-seco-CBI conjugate 5 was similar to that of polyamide-chlorambucil conjugates 1 and 2. In contrast, the cytotoxicity of conjugate 5 was superior to that of conjugates 1-4. These results suggest that the seco-CBI conjugate was distinctly active in cells compared to the chlorambucil conjugates. These results may contribute to the development of more specific and active DNA alkylating agents., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

We designed and synthesized conjugates between pyrrole-imidazole polyamides and seco-CBI that alkylate within the coding regions of the histone H4 genes. DNA alkylating activity on the histone H4 fragment and cellular effects against K562 chronic myelogenous leukemia cells were investigated. One of the conjugates, 5-CBI, showed strong DNA alkylation activity and good sequence specificity on a histone H4 gene fragment. K562 cells treated with 5-CBI down-regulated the histone H4 gene and induced apoptosis efficiently. Global gene expression data revealed that a number of histone H4 genes were down-regulated by 5-CBI treatment. These results suggest that sequence-specific DNA alkylating agents may have the potential of targeting specific genes for cancer chemotherapy., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Sequence-specific DNA alkylating agents have great interest for novel approach to cancer chemotherapy. We designed the conjugates between pyrrole (Py)-imidazole (Im) polyamides and DNA alkylating chlorambucil moiety possessing at different positions. The sequence-specific DNA alkylation by conjugates was investigated by using high-resolution denaturing polyacrylamide gel electrophoresis (PAGE). The results showed that polyamide chlorambucil conjugates alkylate DNA at flanking adenines in recognition sequences of Py-Im polyamides, however, the reactivities and alkylation sites were influenced by the positions of conjugation. In addition, we synthesized conjugate between Py-Im polyamide and another alkylating agent, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). DNA alkylation reactivies by both alkylating polyamides were almost comparable. In contrast, cytotoxicities against cell lines differed greatly. These comparative studies would promote development of appropriate sequence-specific DNA alkylating polyamides against specific cancer cells.

Targeting specific genes or gene products by small molecules is novel approach of cancer chemotherapy. We have developed sequence-specific DNA alkylating agents, conjugates between pyrrole (Py)-imidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H benz[e]indole (seco-CBI) with an indole linker. These compounds efficiently alkylate DNA at a targeted sequence and inhibit gene expression caused by alkylation at template strand of coding region. Recently, histone H4c gene could be targeted by polyamide-chlorambucil (Chl) conjugates. Thus, we designed and synthesized polyamide seco-CBI conjugates 1-5 targeting histone H4c coding sequence. High resolution denaturing polyacrylamide gel electrophoresis (PAGE) showed polyamide seco-CBI conjugates alkylated at the histone H4c coding sequence.

We have developed a series of conjugates between pyrrole (Py)-imidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H benz[e]indole (seco-CBI) with an indole linker. High resolution polyacrylamide gel electrophoresis revealed that these conjugates alkylated DNA at predetermined sequences. Then, we demonstrated that conjugates 1 and 2 have DNA alkylation activities at double stranded human telomere sequence and potent cytotoxicities in cancer cell lines. In addition, we showed that conjugate 3 alkylates DNA with ten-base-pair recognition sequence in the presence of partner polyamide 4, which suggested alkylation through 3-4 heterodimer formation.

We have developed various types of sequence-specific alkylating agents by conjugation of Py-Im polyamides and alkylating moieties 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) with an indole linker. In order to extend the length of target DNA sequence of the hairpin Py-Im polyamide 1, we designed and synthesized Y-shaped and tandem hairpin Py-Im polyamides 2 and 3. High-resolution denaturing polyacrylamide gel electrophoresis using 5'-Texas Red-labeled 465-bp DNA fragments revealed that conjugates 2 and 3 alkylated the adenine of target DNA sequences at nanomolar concentrations. Furthermore, evaluation in human cancer cell lines revealed that these Py-Im polyamides 2 and 3 have strong cytotoxicities.

DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2-6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of -log GI50 (50% growth inhibition concentration) for compounds 1-6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the -log GI50 of compound 1 and its conjugates 2-6 showed extremely low values (R<0). These results suggest that differences in the sequence specificity of DNA alkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis.

We have demonstrated that hairpin pyrrole (Py)- imidazole (Im) polyamide-CBI conjugates selectively alkylate predetermined sequences. In this study, we investigated the effect of alkylation subunits, for example conjugates 1-4 with three types of DNA alkylating units, and Py-Im polyamides with indole linker. Conjugate 3 and 4 selectively alkylated the predetermined sequences as described previously, while conjugates 1 and 2 alkylate at mismatched sites.

We have developed a series of novel DNA alkylating polyamides possessing indole linkers. Investigations using high-resolution gel electrophoresis revealed that the indole linked Py-Im polyamide alkylated at A of a targeted nine base pair matching sequence. Evaluation in human cancer cell lines revealed that the indole linked Py-Im polyamides have strong cytotoxicities. Furthermore, we showed that alkylation of the template strand of the coding region by these polyamides causes effective gene silencing.