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The interaction of novel Pyrrolobenzodiazepine derivatives with DNA

The interaction of novel Pyrrolobenzodiazepine derivatives with DNA
The interaction of novel Pyrrolobenzodiazepine derivatives with DNA
Pyrrolobenzodiazepines (PBDs) are naturally occurring antitumour antibiotics that interact and bind to the minor groove of DNA. They have a three-ring structure containing an anthranilate ring followed by diazepine ring and then a pyrrolidine ring. The diazepine ring contains an imine group that is able to form a covalent bond to guanine in the DNA minor groove. Different modifications to the structure of PBDs can be made in order to alter their binding site size, sequence selectivity and binding affinity. Different modifications include the attachment of groups that interact non-covalently with the DNA minor groove, the formation of PBD dimers or the attachment of other covalent binding minor groove binders.
The work in this thesis describes studies on three such families of PBD-containing compounds. The PBDs in this study were modified so that the five membered pyrrolidine ring is replaced with a six membered piperidine ring, generating compounds that are known as pyridinobenzodiazepines (PDD). DNase I footprinting and fluorescence melting studies were used to examine the sequence selectivity of PDD mono-alkylators that contained a range of attached pyrrole, imidazole and thiazole group. These compounds generally bound covalently to guanines followed by AT-rich sequences.
The second group of compounds were PDD dimers, designed to crosslink two guanines, but also containing additional functional groups in the linker. DNase I footprinting and fluorescent melting experiments showed that these novel PDD dimers attached to two guanines, but also formed an additional non-covalent interaction with a central guanine opposite the linker. At other sites these dimers formed either inter- or intra-strand interactions with guanines that are separated by four base pairs. The formation of inter-strand crosslinks was confirmed by a crosslinking assay. Probing the complexes with diethylpyrocarbonate and potassium permanganate showed that binding of the PDD dimers to the DNA did not cause a large distortion to the helix structure.
The last group of compounds examined were dimers containing a PDD and a cyclopropabenzaindole (CBI). CBI’s are analogues of the naturally occurring compounds duocarmycins that covalently bind to adenine in the DNA minor groove. Cleavage experiments showed that the PDD-CBI conjugates bound to the sequence GXXA. Extending the linker with two extra methyl groups changed the recognition to GXXXA. Addition of the PDD reduced the number of CBI reaction sites, showing that the addition of the PDD affected the sequence selectivity. The crosslinking assay confirmed that the PDD-CBI conjugates were able to form inter-strand crosslinks.
University of Southampton
Di Mascio, Daniella
915ed5fb-e344-42eb-bca6-86b52e5200e8
Di Mascio, Daniella
915ed5fb-e344-42eb-bca6-86b52e5200e8
Fox, Keith
9da5debc-4e45-473e-ab8c-550d1104659f

Di Mascio, Daniella (2022) The interaction of novel Pyrrolobenzodiazepine derivatives with DNA. University of Southampton, Doctoral Thesis, 269pp.

Record type: Thesis (Doctoral)

Abstract

Pyrrolobenzodiazepines (PBDs) are naturally occurring antitumour antibiotics that interact and bind to the minor groove of DNA. They have a three-ring structure containing an anthranilate ring followed by diazepine ring and then a pyrrolidine ring. The diazepine ring contains an imine group that is able to form a covalent bond to guanine in the DNA minor groove. Different modifications to the structure of PBDs can be made in order to alter their binding site size, sequence selectivity and binding affinity. Different modifications include the attachment of groups that interact non-covalently with the DNA minor groove, the formation of PBD dimers or the attachment of other covalent binding minor groove binders.
The work in this thesis describes studies on three such families of PBD-containing compounds. The PBDs in this study were modified so that the five membered pyrrolidine ring is replaced with a six membered piperidine ring, generating compounds that are known as pyridinobenzodiazepines (PDD). DNase I footprinting and fluorescence melting studies were used to examine the sequence selectivity of PDD mono-alkylators that contained a range of attached pyrrole, imidazole and thiazole group. These compounds generally bound covalently to guanines followed by AT-rich sequences.
The second group of compounds were PDD dimers, designed to crosslink two guanines, but also containing additional functional groups in the linker. DNase I footprinting and fluorescent melting experiments showed that these novel PDD dimers attached to two guanines, but also formed an additional non-covalent interaction with a central guanine opposite the linker. At other sites these dimers formed either inter- or intra-strand interactions with guanines that are separated by four base pairs. The formation of inter-strand crosslinks was confirmed by a crosslinking assay. Probing the complexes with diethylpyrocarbonate and potassium permanganate showed that binding of the PDD dimers to the DNA did not cause a large distortion to the helix structure.
The last group of compounds examined were dimers containing a PDD and a cyclopropabenzaindole (CBI). CBI’s are analogues of the naturally occurring compounds duocarmycins that covalently bind to adenine in the DNA minor groove. Cleavage experiments showed that the PDD-CBI conjugates bound to the sequence GXXA. Extending the linker with two extra methyl groups changed the recognition to GXXXA. Addition of the PDD reduced the number of CBI reaction sites, showing that the addition of the PDD affected the sequence selectivity. The crosslinking assay confirmed that the PDD-CBI conjugates were able to form inter-strand crosslinks.

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Published date: 31 May 2022

Identifiers

Local EPrints ID: 468348
URI: http://eprints.soton.ac.uk/id/eprint/468348
PURE UUID: 02b13874-6040-4ccb-b4ae-309a5c937fd4
ORCID for Keith Fox: ORCID iD orcid.org/0000-0002-2925-7315

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Date deposited: 10 Aug 2022 18:16
Last modified: 11 Aug 2022 01:32

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