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Interaction of Pyrrolobenzodiazepine conjugates with DNA

Interaction of Pyrrolobenzodiazepine conjugates with DNA
Interaction of Pyrrolobenzodiazepine conjugates with DNA
Pyrrolobenzodiazepines (PBD) are a group of naturally occurring antitumor antibiotics that consist of a tricyclic system containing anthranilate, diazepine and pyrrolidine ring. The compounds bind preferentially to 5’-Pu-G-Pu-3’ sequences in the minor groove of double stranded DNA by covalently linking the diazepine ring with the 2-amino group of guanine. The PBD binding sites can be extended by conjugating the C8 position of the anthranilate ring with heterocyclic moieties such as pyrrole, imidazole and thiazole. Alternatively, benzofused rings such as benzothiophene, benzofuran and indole can be used for conjugate formation; some natural compounds containing these benzofused heterocycles have been found to show anticancer activities.

We have examined the sequence selectivity of PBD-conjugates using DNase I footprinting with substrates that contain every tetranucleotide (MS1 and MS2) and every symmetrical hexanucleotide (HexA and HexB). Conjugates with pyrrole, imidazole and thiazole were found to bind adjacent to AT-rich sequences; their binding preferences towards AT-rich sequences were confirmed with synthetic DNA fragments in which the compounds bound at ATATAT with a guanine at either the 5’- or 3’-end. Conjugation with benzothiophene and benzofuran benzofused rings produced similar binding profiles, though indole conjugates did not appear to bind. The benzofusedconjugates bound at lower concentrations than the polyamide conjugates. These binding sites indicate that the conjugated component plays a vital role in determining the sequence selectivity, while the PBD unit forms the covalent linkage with guanine. PBD-conjugates were also studied by fluorescence melting to evaluate their effect on melting profiles of different oligonucleotide duplexes; the compounds produced biphasic melting curves with large increases in melting temperature.

We also describe a simple ligation assay for determining the sequence selectivity of PBD-dimers. The assay involves chemically joining two DNA duplexes by a PBD-dimer when a binding site is generated by overhanging single-stranded ‘sticky ends’. Oligonucleotides containing GATC or GTAC or GAATC overhangs were used to demonstrate that SJG-136 has strongest affinity for GATC; it also produces cross-links with GTAC, but not with GAATC.

The final chapter uses DNase I footprinting to examine the sequence specificity of two pyrrole/imidazole hairpin polyamides that were expected to bind to the sequences (A/T)GG(A/T)G(A/T) and (A/T)GG(A/T)C(A/T). We confirm the expected selectivity, but demonstrate that they can also form strong interactions with some secondary sites.
Basher, Mohammad Anwarwul
936bf43e-dc1f-49a7-b445-65628e92d868
Basher, Mohammad Anwarwul
936bf43e-dc1f-49a7-b445-65628e92d868
Fox, Keith
9da5debc-4e45-473e-ab8c-550d1104659f

Basher, Mohammad Anwarwul (2015) Interaction of Pyrrolobenzodiazepine conjugates with DNA. University of Southampton, Centre for Biological Sciences, Doctoral Thesis, 320pp.

Record type: Thesis (Doctoral)

Abstract

Pyrrolobenzodiazepines (PBD) are a group of naturally occurring antitumor antibiotics that consist of a tricyclic system containing anthranilate, diazepine and pyrrolidine ring. The compounds bind preferentially to 5’-Pu-G-Pu-3’ sequences in the minor groove of double stranded DNA by covalently linking the diazepine ring with the 2-amino group of guanine. The PBD binding sites can be extended by conjugating the C8 position of the anthranilate ring with heterocyclic moieties such as pyrrole, imidazole and thiazole. Alternatively, benzofused rings such as benzothiophene, benzofuran and indole can be used for conjugate formation; some natural compounds containing these benzofused heterocycles have been found to show anticancer activities.

We have examined the sequence selectivity of PBD-conjugates using DNase I footprinting with substrates that contain every tetranucleotide (MS1 and MS2) and every symmetrical hexanucleotide (HexA and HexB). Conjugates with pyrrole, imidazole and thiazole were found to bind adjacent to AT-rich sequences; their binding preferences towards AT-rich sequences were confirmed with synthetic DNA fragments in which the compounds bound at ATATAT with a guanine at either the 5’- or 3’-end. Conjugation with benzothiophene and benzofuran benzofused rings produced similar binding profiles, though indole conjugates did not appear to bind. The benzofusedconjugates bound at lower concentrations than the polyamide conjugates. These binding sites indicate that the conjugated component plays a vital role in determining the sequence selectivity, while the PBD unit forms the covalent linkage with guanine. PBD-conjugates were also studied by fluorescence melting to evaluate their effect on melting profiles of different oligonucleotide duplexes; the compounds produced biphasic melting curves with large increases in melting temperature.

We also describe a simple ligation assay for determining the sequence selectivity of PBD-dimers. The assay involves chemically joining two DNA duplexes by a PBD-dimer when a binding site is generated by overhanging single-stranded ‘sticky ends’. Oligonucleotides containing GATC or GTAC or GAATC overhangs were used to demonstrate that SJG-136 has strongest affinity for GATC; it also produces cross-links with GTAC, but not with GAATC.

The final chapter uses DNase I footprinting to examine the sequence specificity of two pyrrole/imidazole hairpin polyamides that were expected to bind to the sequences (A/T)GG(A/T)G(A/T) and (A/T)GG(A/T)C(A/T). We confirm the expected selectivity, but demonstrate that they can also form strong interactions with some secondary sites.

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More information

Published date: 29 September 2015
Organisations: University of Southampton, Centre for Biological Sciences

Identifiers

Local EPrints ID: 386977
URI: https://eprints.soton.ac.uk/id/eprint/386977
PURE UUID: 10b43a4f-7952-49f6-b57d-4f96e1f0b565
ORCID for Keith Fox: ORCID iD orcid.org/0000-0002-2925-7315

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Date deposited: 12 Feb 2016 16:56
Last modified: 06 Jun 2018 13:16

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