STRUCTURAL STUDIES OF HUMAN TOPOISOMERASE I AND DRUG PROCESSING ESTERASES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The examination of two proteins of importance in human disease is proposed. First, topoisomerase I plays a critical role in DNA metabolism by relaxation superhelical tension and is also the sole target of the anti-cancer drug camptothecin. The P.I. of this application determined crystal structures of human topoisomerase I in complexes with DNA using the facilities at SSRL. We now propose to examine camptothecin resistant and hypersensitive forms of the enzyme in complex with DNA to unravel the mode of action of these drugs. Crystals of this protein-DNA complex are in-hand but diffract x-ray weakly in-house (~4.5 ¿); thus synchrotron radiation is required. Second, the camptothecin derivative Irinotecan is processed in vivo to its active form by liver carboxylesterases. We have crystallized a rabbit liver carboxylesterase that efficiently activates Irinotecan. The goal of these studies is to understand how this critical cancer drug is processed in humans. Hexagonal crystals of this enzyme (space group P6322) diffract x-rays to 3.8 ¿ in-house and contain a long c-axis (275 ¿). Synchrotron radiation is required to improve diffraction resolution and to separate reflections that overlap due to geometric constraints at our home source.