Analysis of human topoisomerase I inhibition and interaction with the cleavage site +1 deoxyguanosine, via in vitro experiments and molecular modeling studies

Laco, GS and Du, W and Kohlhagen, G and Sayer, JM and Jerina, DM and Burke, TG and Curran, DP and Pommier, Y (2004) Analysis of human topoisomerase I inhibition and interaction with the cleavage site +1 deoxyguanosine, via in vitro experiments and molecular modeling studies. Bioorganic and Medicinal Chemistry, 12 (19). 5225 - 5235. ISSN 0968-0896

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Abstract

Human topoisomerase I Tyr723 (white) in covalent complex with DNA (light blue) via the -1 deoxythymidine (bottom), while the +1 deoxyguanosine with a free 5′-OH is rotated out of the helix (right). Homocamptothecin 9,10 diF (green) is docked in the active site (A-E rings, left to right). Active-site residues (yellow) that make H-bond/electrostatic contacts with either inhibitor, or the rotated +1 deoxyguanosine, are shown. Oxygen, red; nitrogen, blue; fluorine, magenta. Human topoisomerase I (Top1) plays a pivotal role in cell replication and transcription, and therefore is an important anti-cancer target. Homocamptothecin is a lead compound for inhibiting Top1, and is composed of five conjugated planar rings (A-E). The homocamptothecin E-ring β-hydroxylactone opens slowly to a carboxylate at pH < 7.0. We analyzed, which form of homocamptothecin was biochemically relevant in the following ways: (1) the homocamptothecin carboxylate was tested for activity in vitro and found to be inactive; (2) homocamptothecin was incubated with Top1 and dsDNA, and we found that the homocamptothecin β-hydroxylactone form was stabilized; (3) the homocamptothecin E-ring β-hydroxylactone was modified to prevent opening, and the derivatives were either inactive or had low activity. These results indicated that the homocamptothecin β- hydroxylactone was the active form, and that an E-ring carbonyl oxygen and adjacent unsubstituted/unprotonated ring atom were required for full activity. Homocamptothecin and derivatives were docked into a Top1/DNA active site model, in which the +1 deoxyguanosine was rotated out of the helix, in order to compare the interaction energies between the ligands and the Top1/DNA active site with the in vitro activities of the ligands. It was found that the ligand interaction energies and in vitro activities were correlated, while the orientations of the ligands in the Top1/DNA active site explained the importance of the E-ring β-hydroxylactone independently of E-ring opening. An essential component of this Top1/DNA active site model is the rotated +1 deoxyguanosine, and in vitro experiments and molecular modeling studies supported rotation of the +1 deoxyguanosine out of the helix. These results allow for the rational design of more potent Top1 inhibitors through engineered interactions with as yet unutilized Top1 active-site residues including: Glu356, Asn430, and Lys751. © 2004 Elsevier Ltd. All rights reserved.

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Details

Item Type:	Article
Status:	Published
Creators/Authors:	Creators Email Pitt Username ORCID Laco, GS Du, W Kohlhagen, G Sayer, JM Jerina, DM Burke, TG Curran, DP curran@pitt.edu CURRAN Pommier, Y
Date:	1 October 2004
Date Type:	Publication
Journal or Publication Title:	Bioorganic and Medicinal Chemistry
Volume:	12
Number:	19
Page Range:	5225 - 5235
DOI or Unique Handle:	10.1016/j.bmc.2004.06.046
Schools and Programs:	Dietrich School of Arts and Sciences > Chemistry
Refereed:	Yes
ISSN:	0968-0896
MeSH Headings:	Binding Sites; Camptothecin--analogs & derivatives; DNA; DNA Topoisomerases, Type I--chemistry; Deoxyguanosine--chemistry; Humans; Hydrophobic and Hydrophilic Interactions; Ligands; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Static Electricity; Thermodynamics; Topoisomerase I Inhibitors
PubMed ID:	15351405
Date Deposited:	07 Aug 2013 15:01
Last Modified:	29 Jan 2019 15:55
URI:	http://d-scholarship-dev.library.pitt.edu/id/eprint/19576

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