Book of Abstracts: Albany 2003
June 17-21 2003
How Camptothecin Inhibits Human Topoisomerase I and How it forms an Alkali-Labile Complex with DNA (The Models)
In mid-1980s Fukada (1) described formation of a camptothecin (CPT) complex with circular supercoiled DNA molecules in the absence of DNA topoisomerase I (topo I) and called it alkali-labile. This term was due to the fact that sedimentation of this complex under alkaline conditions released nicked DNA molecules. Previously we proposed a model for a complex of the topothecan (TPT, a derivative of CPT) lactone dimer with two intersecting DNA duplexes in the absence of topo I (2). The model suggests that the carbonyl group at the C16a atom forms a hydrogen bond with the 2-amino group of guanine, in this case the lactone oxygen is at a distance of ≡4 Å from the phosphate group at the 3? end of this guanine. On the other hand, it is known that the DNA-bound CPT molecules undergo the lactone?carboxylate transition (3).
Based on these two concepts, we proposed a model for interaction between the CPT carboxylate form and DNA in the absence of topo I (4). Other authors (5) also admit the possibility of the TPT carboxylate interaction with DNA. According to our model, the CPT in lactone form binds to DNA, and after transition to carboxylate form while bound to DNA, it induces a nick in the sugar-phosphate backbone. In this case the hydroxyl group emerging at C17 forms a covalent bond with the phosphate group at one end of the break, whereas the carboxyl appearing at C20 forms two additional hydrogen bonds, one with the N1 atom and the other by the other proton with the same 2-amino group of guanine, which is at the other end of the break. The resulting complex is stable at neutral pH. It is destroyed only under alkaline conditions, probably when the phosphate group switches from the hydroxyl at C17 to a hydroxyl of water. The structure of the proposed alkali-labile complex is shown in Figure 1 (hydrogen bonds are shown by a dotted line).
The model was extended to the topo I inhibition by CPT. At the first step, CPT in lactone form also binds to DNA. After phosphate transfer to the hydroxyl of Tyr723 (topo I) and CPT transition into the carboxylate form, the second step of their interaction begins. The CPT carboxylate establishes an additional bifurcation hydrogen bond from the hydroxyl at its C17 to the N1 and N3 of the same guanine, whereas the carboxyl at C20 forms hydrogen bonds with NH of Tyr723 and Nδ2H2 of adjacent Asn722. Guanine is at one end of the break while Tyr723 is covalently bound to the other end. Moreover, rotation of one DNA strand about the other, which is necessary to shed one supercoil, becomes impossible, and formation by guanine of additional hydrogen bonds probably alters its position in space so that re-ligation (without supercoil shedding) is also precluded. Just this explains the inhibitory effect of the CPT family agents on topo I.
The work was partially supported by the Russian Foundation for Basic Research (grants 01-04-48657, 01-03-32669).
Sergei A. Streltsov
Engelhardt Institute of Molecular Biology