1. Field of the Invention
The present disclosure relates to a crystal structure of a protein-DNA binary complex. More particularly, the disclosure relates to the crystal structure of a human topoisomerase II (hTOPII)-DNA binary complex and its uses for identifying candidate compounds exhibiting inhibitory effects toward hTOPII, specifically, the cc and (3 form of hTOPII.
2. Description of Related Art
DNA topoisomerases (TOPs) are enzymes that change DNA topology by catalyzing the passage of DNA strands across each other. There are two well-characterized classes of topoisomerases, type I topoisomerase (TOPI) and type II topoisomerase (TOPII). TOPI acts by breaking and religating one DNA strand, while TOPII involves double-strand breaking. These enzymes play key roles in DNA replication, transcription, and recombinant repair. In particular, TOPII is highly expressed in rapidly proliferating cells and is therefore an attractive target for the development of antitumor drugs.
TOPIIs are two-fold symmetric enzymes that alter DNA topology by transiently cleaving a pair of opposing phosphodiester bonds four base pairs apart via the formation of two reversible 5′-phosphotyrosyl linkages, thereby generating a TOPII-DNA cleavage complex that harbors a double-stranded DNA break. Under physiological conditions, these TOPII-DNA cleavage complexes are normally short lived intermediates in the catalytic cycle of the enzyme. Passage of a second DNA segment through this TOPII-DNA cleavage complex and its resealing complete the topological change of the DNA. The DNA cleavage activity of TOPII is known as a double-edged sword; failure to reseal the enzyme-mediated DNA break can lead to permanent DNA damage followed by cell death.
There are two general classes of TOPII-targeting drugs: TOPII poisons and TOPII catalytic inhibitors. TOP II poisons include etoposide (VP-16), doxorubicin, amsacrine (mAMSA) and mitoxantrone. These compounds serve to stabilize the TOPII-DNA cleavage complex thereby increasing the steady-state levels of cleavage complexes. Hence TOPII poisons convert TOPII into a physiological toxin that creates DNA double-strand breaks in the genome of treated cells. The catalytic inhibitors, on the other hand, block the catalytic activity of DNA-TOPII cleavage complex by preventing the binding of the enzyme to DNA rather than stabilizing the DNA-TOPII cleavage complex. Examples of catalytic TOPII inhibitors include novobiocin, dexrazoxane (ICRF-187) and GSK 299423.
Crystallography is a powerful tool for investigating the interactions among protein, DNA, and drugs. In this disclosure, crystal structures of the DNA-binding and cleavage core of hTOPII (hTOPII core domain; hTOPIIcore) in complexes with DNA and antitumor drugs were used to obtain high resolution and homogeneous crystal structure of binary TOPIIcore-DNA cleavage complex, which is useful for constructing a high throughput screening platform for identifying candidate compounds having inhibitory effects toward hTOPII. As such, potential lead compound having anti-tumor effects may be identified and subsequently being developed into an anti-neoplastic medicine.