Sequence Specific Trapping of Topoisomerase I by Camptothecin Polyamide Conjugates

Author: Wang, Clay Chia Chun

Year: 2002

Degree: Dissertation (Ph.D.)

Advisors: Readhead, Anthony C. S.; Dervan, Peter B.

Committee Members: Rees, Douglas C.; Readhead, Anthony C. S.; Dervan, Peter B.; Stoltz, Brian M.; Roberts, Richard W.

Option: Chemistry

DOI: 10.7907/sphn-ma74

Abstract

Polyamides containing N-methylpyrrole (Py)N-methylimidazole (Im) and 3-hydroxy-1-methylpyrrole (Hp) are cell permeable small molecules that bind as antiparallel pairs in the minor groove of DNA according to a set of "pairing rules" with affinities and specificities for predetermined sequences comparable to DNA-binding proteins. Several of these ring pairings have only been demonstrated when placed internal in a hairpin and not for the terminal position in hairpin polyamides. Several series of eight ring hairpin polyamides with modification at the N-terminal position were synthesized and characterized. We observed that at the terminal position (i) the Py/Py pair functions similar to when placed internally preferring A•T and T•A base pairs over O•C and C•O, (ii) the Hp/Py pair could not distinguish between T•A over A-T possibly due to rotation of the terminal Hp to form an intramolecular hydrogen bond between the 3-hydroxyl hydrogen and the carboxamide oxygen which would orient the key hydroxyl recognition element away from the minor groove. A new aromatic pair, 2-hydroxy-6-methoxybenzamidell-methylpyrrole was designed and shown to distinguish T•A from A•T base pairs and both from O•C/C•O and (iii) the Py/Im pair in the classic eight ring hairpin motif showed no preference for C•O base pair possibly due to the mispositioning of the Im residue located at the C-terminal end of the four ring polyamide subunit. Targeting of C•O was accomplished by replacing a pyrrole with a flexible β-alanine and setting the imidazole back in register.

Pyrrole-imidazole polyamides that target DNA sequences in the promoter have been shown to inhibit transcription of specific genes in cell culture. When bound to coding region of genes, polyamides do not appear to inhibit gene expression. A possible solution is to design molecule capable of modifying DNA when bound to the coding region. A series of polyamide-camptothecin conjugates were designed to trap the enzyme Topoisomerase I and induce cleavage at predetermined DNA sites. Cleavage yields were shown to be dependent on linker length between the DNA binding polyamide and the Topo I trapping camptothecin unit with the camptothecin unit with the longest linker showing greatest cleavage yield of over 90%.

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