Biomedical subjects

Toshikazu Bando

Publications and source records attributed to Toshikazu Bando.

29 records · Page 2Linked to original sources

PubMed2003

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.

Adenine↗

PubMed2003

Inhibition of transcription at a coding sequence by alkylating polyamide.

Transcription from DNA sequence-specifically alkylated by a hairpin polyamide (ImPyPy-gamma-ImPyLDu86, 1) was investigated. High-resolution polyacrylamide gel electrophoresis demonstrated that conjugate 1 alkylated a 993-bp DNA fragment, in accordance with the Py-Im recognition rule, predominantly at the one match site on the GFP-encoding strand and at four sites (I'-IV') on the noncoding strand. Alkylation of DNA inhibited the formation of full-length mRNA and caused the transcription of truncated mRNA. Polyacrylamide gel electrophoresis demonstrated that the length of the truncated mRNA was consistent with the alkylated site on the coding strand. Complete inhibition of full-length mRNA synthesis was observed in the presence of 50 nM 1. In clear contrast, the hydrolyzed derivative of 1, designated 2, produced no truncated mRNA, nor did it significantly retard transcription: >80% transcription of full-length mRNA was observed at 50 nM. These results clearly indicate that inhibition of transcription can be achieved with alkylating Py-Im polyamide even in the coding regions of genes.

Alkylation↗

PubMed2003

Highly efficient sequence-specific DNA interstrand cross-linking by pyrrole/imidazole CPI conjugates.

We have developed a novel type of DNA interstrand cross-linking agent by synthesizing dimers of a pyrrole (Py)/imidazole (Im)-diamide-CPI conjugate, ImPyLDu86 (1), connected using seven different linkers. The tetramethylene linker compound, 7b, efficiently produces DNA interstrand cross-links at the nine-base-pair sequence, 5'-PyGGC(T/A)GCCPu-3', only in the presence of a partner triamide, ImImPy. For efficient cross-linking by 7b with ImImPy, one A.T base pair between two recognition sites was required to accommodate the linker region. Elimination of the A.T base pair and insertion of an additional A.T base pair and substitution with a G.C base pair significantly reduced the degree of cross-linking. The sequence specificity of the interstrand cross-linking by 7b was also examined in the presence of various triamides. The presence of ImImIm slightly reduced the formation of a cross-linked product compared to ImImPy. The mismatch partners, ImPyPy and PyImPy, did not produce an interstrand cross-link product with 7b, whereas ImPyPy and PyImPy induced efficient alkylation at their matching site with 7b. The interstrand cross-linking abilities of 7b were further examined using denaturing polyacrylamide gel electrophoresis with 5'-Texas Red-labeled 400- and 67-bp DNA fragments. The sequencing gel analysis of the 400-bp DNA fragment with ImImPy demonstrated that 7b alkylates several sites on the top and bottom strands, including one interstrand cross-linking match site, 5'-PyGGC(T/A)GCCPu-3'. To obtain direct evidence of interstrand cross-linkages on longer DNA fragments, a simple method using biotin-labeled complementary strands was developed, which produced a band corresponding to the interstrand cross-linked site on both top and bottom strands. Densitometric analysis indicated that the contribution of the interstrand cross-link in the observed alkylation bands was approximately 40%. This compound efficiently cross-linked both strands at the target sequence. The present system consisted of a 1:2 complex of the alkylating agent and its partner ImImPy and caused an interstrand cross-linking in a sequence-specific fashion according to the base-pair recognition rule of Py-Im polyamides.

Base Sequence↗

PubMed2003

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.

Alkylation↗

PubMed2003

Molecular design of hairpin pyrrole-imidazole polyamides possessing sequence specific DNA alkylating moiety.

By conjugating pyrrole (Py)/imidazole (Im) hairpin polyamides with CBI, a stable alkylating moiety, we have developed a new type of DNA-sequence-specific alkylating agent. The sequence specificity and alkylation efficiency of the CBI conjugates were analyzed with high-resolution denaturing gel electrophoresis using linear 450- and 1000-bp DNA. The results demonstrate that CBI conjugates selectively alkylate adenine in specific sequences, whereas the previous type of alkylating polyamide, which contains segment A of DU-86, alkylates both adenine and guanine.

Alkylation↗

PubMed2003

Fmoc solid-phase synthesis and its application to pyrrole-imidazole polyamides.

Improvements to the Fmoc solid-phase synthesis of N-methylpyrrole (Py) and N-methylimidazole (Im) polyamides were examined. It was found that a different combination of coupling agents and activators, and the introduction of a dimer unit significantly enhanced the yield of polyamides. Using this improved method, various types of polyamides were synthesized. The uptake and localization of polyamides in living mammalian cells were investigated using FITC labeled Py-Im polyamides.

Chromatography, High Pressure Liquid↗

PubMed2002

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 Dervan's 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.

Alkylation↗

PubMed2002

Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates.

The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 specifically alkylated DNA at 5'-CGCGCG-3' and 5'-PyGGCCPu-3', respectively. Agarose gel electrophoresis indicated that incubation of a supercoiled plasmid, pSPORT I (4109 bp), with conjugate 7 effectively inhibited its hydrolysis by BssHII (5'-G_CGCGC-3'), whereas conjugate 14 had no effect on this hydrolysis. These results suggest that conjugate 7 sequence-specifically inhibits the hydrolysis of DNA by BssHII. Sequence-specific alkylation by the Py-Im triamide-CPI conjugates was further confirmed by inhibition of the Eco52I (5'-C_GGCCG-3') hydrolysis of conjugate 14-treated pQBI PGK (5387 bp). In clear contrast, hydrolysis of pQB1 PGK by DraI (3'-TTT_AAA-3') was not inhibited by 5 micro M conjugate 14. That ImImPy did not inhibit the hydrolysis of pQB1 PGK indicates that covalent bond formation is necessary for inhibition. A similar experiment, using linear pQBI PGK, achieved the same extent of protection of the DNA with approximately half the concentration of conjugate 14 as was required to protect supercoiled DNA from hydrolysis.

Alkylation↗

PubMed2002

Molecular design of hairpin polyamide possessing sequence-specific alkylation activity.

A novel type of sequence specific hairpin polyamide alkylating agents were synthesized and their alkylation activities were evaluated on linear 450 bp DNA fragment. To optimize the design of hairpin polyamide CPI-conjugates, several structural analogues were prepared. Analysis using high resolution denaturing gel electrophoresis suggests that such subtle structural differences effect the sequence specificity of the polyamides.

Alkylating Agents↗

PubMed2002

Regulation of gene expression by sequence-specific alkylating polyamide.

In order to investigate the inhibition of gene expression by a new type of hairpin polyamide-CPI conjugate 1, its ability to inhibit transcription in cell free system was investigated. Sequence-selective alkylation of double-stranded DNA by 1 was investigated by denaturing gel electrophoresis using 1000 bp DNA fragment which codes for green fluorescence protein (GFP) under the control of T7 promoter. Analysis of DNA sequence indicated that 1 alkylated predominantly at the site of 5'-AGTCA-3' in coding region of GFP. The transcript by T7 RNA polymerase using the alkylated DNA as a template was analyzed by PAGE. The results clearly indicate that 1 inhibits transcription by alkylation of coding region at a nanomolar concentration.

Alkylation↗

PubMedDate not supplied

Efficient DNA alkylation by a pyrrole-imidazole CBI conjugate with an indole linker: sequence-specific alkylation with nine-base-pair recognition.

Conjugates 7, 8, and 10 of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) with a 5-amino-1H-indole-2-carbonyl linker were synthesized by Fmoc solid-phase synthesis and a subsequent liquid-phase coupling procedure. The DNA alkylating abilities of conjugates 7, 8, 6b, and 10 were examined using Texas Red-labeled PCR fragments and high-resolution denaturing gel electrophoresis. CBI conjugates 7 and 8 exhibited highly efficient sequence-specific DNA alkylation comparable with previous CBI conjugates with a vinyl linker. In particular, conjugate 10, with a 10-ringed hairpin Py-Im polyamide, alkylated at the adenine of 5'-ACAAATCCA-3'. Introduction of an indole linker greatly facilitated the synthesis of sequence-specific alkylating Py-Im polyamides.

Alkylation↗