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MODELING TOPOISOMERASE-DNA INTERACTIONS AND DESIGN of TRAPPING INHIBITORS USED FOR CANCER

 by

Soaring Bear

 ___________________

Copyright © Soaring Bear 1998

all rights reserved

 

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF PHARMACEUTICAL SCIENCES

 

In Partial Fulfillment of the Requirements

 

For the Degree of PhD

 

With a Major in Pharmaceutical Sciences

and a Minor in Biochemistry

In the Graduate College

The University of Arizona, 1998

  

TABLE OF CONTENTS

ABSTRACT

INTRODUCTION

Cancer touches all of us

Topoisomerase targeting of chemotherapy

Three types of topoisomerase in humans

Oncogenes near topoisomerase genes help targeting

Targeting high levels of topoisomerase 2

How inhibition of topoisomerase 2 kills cells

Inhibition of topoisomerase

Designing better topoisomerase trapping agents

Intercalation is insufficient

Trans-esterification

Kinetics of topoisomerase winding of DNA

Categorizing topoisomerase inhibitors

Amonafide & Azonafide background

STRUCTURE METHODS

STRUCTURE RESULTS AND DISCUSSION

Sequence analysis of human topoisomerase

Yeast structure insights

Overhang 5’ or 3’?

Drug selected mutant bioinformatic analysis

Homology of human topoisomerase structure from yeast

Active Site Analysis

INHIBITOR METHODS

INHIBITOR RESULTS AND DISCUSSION

Error in force field based calculations

SAR of topoisomerase inhibitors

Conclusions, implications and suggestion for future study

REFERENCES

  

ABSTRACT

Topoisomerase inhibiting chemotherapy is most appropriate in cancers containing high levels of topoisomerase. Study of these drugs is particularly difficult because two receptors, enzyme plus DNA, are involved. Genes of all three known human topoisomerases have neighboring oncogenes and those amplified receptors may be used to target topoisomerase inhibitors to transformed cells. To facilitate design of inhibitors, modeling tools of graphic imaging, sequence analysis, homology, molecular dynamics, energy evaluation, and QSAR were used. The principle findings are:

  1. The widely accepted model of 5’ attachment to DNA and 5’ overhang is inconsistent with topoisomerase structure; 3’ overhang is more likely.
  2. The active site region near the catalytic tyrosine hydroxyl is well conserved between yeast and humans with some exceptions: 4 A away Glu554->His566 is likely to modify the proton transport of the trans-esterification reaction conducted by this enzyme; two base pairs away, the yeast Asp 552 appears to probe DNA major groove and becomes oppositely charged Arg 565 in humans; 8-15 A away along the probable location of DNA backbone, the acid pair Glu-Asp 512-13 is conserved and repeated in humans as Glu-Asp-Glu-Asp (522-5); these residues might coordinate the required Mg2+ along with the conserved Asp 635 (644 in human); the acidic triad 590-2 evolves to a hydroxy triad and might provide a proton transport pathway for the trans-esterification reaction.
  3. Drug resistant mutant structure analysis suggests multiple sites of action on topoisomerase and suggests the region of DNA binding.
  4. Prediction of intercalator binding to DNA is correctly ranked by molecular mechanics for amonafide and azonafide; however, there remains significant error from experimental measures; force field error derives from the approximation of electron orbitals as van der Waals sphere, lack of polarization function, poor handling of hydrogen bond variability, and force field parameterization from packed protein crystals.
  5. Wide variability of the azonafide analogs on whole cell assays makes QSAR difficult and suggests other biochemical pathways are affected; the measurements need to be redone on a simple enzyme-DNA assay.