Camptothecin (CPT) is a pentacyclic alkaloid that is isolated from the Chinese tree Camptotheca acuminata.  It is used in the preparation of anticancer drugs, such as Topotecan and Irinotecan.  These drugs belong to a new class of anticancer agents that act to inhibit topoisomerase I.
 
 

Camptothecin

Topotecan	Irinotecan

Mechanism of Topoisomerase I

        Toposimerase I is an enzyme used during transcription and DNA replication to relax any supercoiled DNA.  It does so by causing a single-strand break in the DNA.  The tyrosine-723 residue of the enzyme active site acts as a nucleophile and breaks the phosphodiester bond of the DNA strand.  This creates a 3'-phophotyrosol linkage between the enzyme and the broken DNA strand.  After the DNA is relaxed, the free 5'-hydroxyl group attacks the phosphotyrosine intermediate, which religates the ends of the broken DNA strand.
 
 
 

Mechanism of Topoisomerase I Inhibitors

        Camptothecin and its analogues act as uncompetative inhibitors and bind the 3' phosphotyrosine intermediate, blocking the religation of the DNA strands.  This "traps" the topoisomerase I on the DNA, so that during DNA replication, the replication fork collides with the complex.  The collision causes double-stranded breakage of the DNA and eventually cell death.

        Wang et al reported the x-ray crystal structure of this ternary complex with Topotecan and have shown that the planar five-membered rings of Topotecan mimic a DNA base pair.  The Topotecan intercalates at the DNA cleavage site and forms base-stacking interactions with the upstream (-1) and downstream (+1) DNA base pairs.  This intercalation displaces the free 5'-hydroxyl group of the cleaved DNA strand further away from the 3'-phosphotyrosol linkage.
 
 
 
 

Spin off/on Zoom Side view Lactone/Carboxylate      forms of Topotecan		Arg 364 Asp 533 Asn 722 Phe 361 Gly 363 Glu 418 Ala 653 Thr 729
	Ternary Complex Between Topoisomerase I, DNA, Topotecan

 
 

The residues listed above are important in the mechanism for topoisomerase I inhibitors.  Mutations in these residues result in CPT resistance in tumor cells:

    Arg 364, asp 533, and asn 722 directly contribute to the binding of Topotecan within the topoisomerase I-DNA complex.  Phe 361 and gly 363 do not directly contact Topotecan, but help to create and stabilize the intercalation binding pocket.  They hydrogen bond to the upstream and downstream base pairs of the uncleaved DNA strand to help open the DNA duplex.  Glu 418, ala 653, and thr 729 also are not able to directly contact Topotecan.  It is not known what effect these residues have on the structure and stability of the Topotecan binding pocket.
 
 
 
 

References

Protein Data Bank: 1K4T (code for crystal structure of the ternary complex)

Staker, B. L.; Hjerrild, K; Feese, M. D.; Behnke, C. A.; Burgin, A. B.; Stewart, L. The Mechanism of Topoisomerase I Poisoning by a Camptothecin Analogue.  PNAS  2002, 99 (24), 15387-15392.

Wang, X.; Zhou, X.; Hecht, S. M.  Role of the 20-Hydroxyl Group in Camptothecin Binding by the Topoisomerase I-DNA Binary Complex.  Biochemistry  1999, 38, 4374-4381.
 
 

Web Page Author:  Jennifer Daugherty