Probing the interaction of the cytotoxic bisdioxopiperazine ICRF-193 with the closed enzyme clamp of human topoisomerase IIalpha

• Published in: Molecular pharmacology 2000-09, Vol.58 (3), p.560-568
• Main Authors: Patel, S, Jazrawi, E, Creighton, A M, Austin, C A, Fisher, L M
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - drug effects; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amino Acid Substitution; Antigens, Neoplasm; Antineoplastic Agents, Phytogenic - pharmacology; Catalysis; DNA - drug effects; DNA - metabolism; DNA Topoisomerases, Type II - chemistry; DNA Topoisomerases, Type II - genetics; DNA Topoisomerases, Type II - metabolism; DNA-Binding Proteins; Drug Resistance; Enzyme Inhibitors - pharmacology; Etoposide - pharmacology; Humans; Isoenzymes - antagonists & inhibitors; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; Molecular Sequence Data; Multienzyme Complexes - antagonists & inhibitors; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Mutation; Piperazines - pharmacology; Protein Conformation; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - genetics; Sequence Homology, Amino Acid; Topoisomerase II Inhibitors
• ISSN: 0026-895X

Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregation at mitosis. A number of cytotoxic bisdioxopiperazines such as ICRF-193 target topoisomerase II by binding and trapping the closed enzyme clamp. To investigate this unusual mode of action, we have used yeast to select plasmid-borne human topoisomerase IIalpha alleles resistant to ICRF-193. Mutations in topoisomerase IIalpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identified as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, respectively. The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoisomerase and ATPase activities and whose closed clamp was refractory to ICRF-193-mediated trapping as an annulus on closed circular DNA. These data imply that the mutation interferes directly with ICRF-193 binding to the N-terminal ATPase gate. In contrast, the A648P enzyme displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193. We suggest that the inefficient trapping of the A648P closed clamp results either from the observed increased ATP requirement, or more likely, from lowered salt stability, perhaps involving destabilization of ICRF-193 interactions with the B'-B' interface in the core domain. These results provide evidence for at least two different phenotypic classes of ICRF-193 resistance mutations and suggest that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase IIalpha.