1. A Natural Anticancer Agent Thaspine Targets Human
Topoisomerase IB
Supervisor By…
Prof. Alessandro Desideri Prafulla Katkar

2. Topoisomerases are the key enzymes that control the topological state of DNA through the
breaking and rejoining of DNA strands.
Two kinds of Topoisomerases : Type I (IA, IB) and Type II
These enzymes are involved in many vital process such as DNA replication, transcription,
translation and chromosomal segregation etc.

3. Most anticancer drugs have their topoisomerase as their molecular target.
1. Topo poisons : Stabilizes Topo-DNA cleavable complex and inhibit religation.
(Camptothecin and its derivatives topothecan, Gematecan approved in clinical
trials)
2. Topo Inhibitors : Inhibit cleavage of DNA
Cell Death (Apoptosis)

4. Topoisomerase IB Topo poison

5. Dragon’s Blood (Sangre di grado)
An alkaloid present in the cortex of south American tree Croton lechleri
Being used as traditional medicine for several diseases.
Thaspine been known to exhibit promising anticancer properties.
(Stig Linder et al 2009)
1. Has strong cytotoxic effect on multicellular spheroids and induce
apoptosis in human xenograft in vivo.
2. by intercalation of the planar complexes between the DNA
base pairs.
3. and/or by inhibition of enzyme activities.

6. 1. Investigation of Mechanism of effect of Thaspine on Topoisomerase
IB whether it is Topo poison or inhibitor.
2. Analyzing its effect on different steps of enzyme catalysis.

7. Effect of Thaspine on topoisomerase IB relaxation :
Thaspine
C 0 5 10 15 20 50 µM
NC
Relaxed
SC
1 2 3 4 5 6 7
Thaspine inhibits the human topoisomerase IB
in a dose dependent manner

8. Pre-incubation
DMSO Thaspine Thaspine+E
C 1 7.5 15 30 1 7.5 15 30 1 7.5 15 30 Time (Min)
NC
Relaxed
SC
1 2 3 4 5 6 7 8 9 10 11 12 13
Pre-incubation of drug increase the inhibitory effect for short time
Then decreases gradually

9. Pre-incubation
DMSO Thaspine+E
C 1:1 1:2 1:4 1:8 1:1 1:2 1:4 1:8
NC
Relaxed
SC
1 2 3 4 5 6 7 8 9
The inhibitory effect of thaspine is reversible, as the effect is lost as the
dilution increases

10. Pre-incubation
DMSO Thaspine CPT Taspine+E
C 5 15 30 5 15 30 5 15 30 5 15 30
Religated oligo
Cleavable Complex
1 2 3 4 5 6 7 8 9 10 11 12 13
Either thaspine inhibits the cleavage or induces a faster religation,
so that the cleavable complex cannot be formed.

11. DMSO Thaspine
C 0 0.5 1 5 10 20 30 0 0.5 1 5 10 20 30
----- Tasp
DMSO
1 2 3 4 5 6 7 8 9 10 11 1213 14 15
The religation rate is slowed down in presence of thaspine as compared with DMSO

12. Pre-incubation
DMSO Thaspine Thaspine+E
C 0.5 1 5 10 20 60 0.5 1 5 10 20 60 0.5 1 5 10 20 60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
The ability of topoisomerase to cut the DNA is reduced in presence
of Thaspine and the effect is more intense when the thaspine pre-incubated
with enzyme.

13. Thaspine binds to enzyme with good interaction energy, before its
interaction with DNA

14. A. The intercalation of thaspine between the bases of DNA, evidence
for inhibition of religation.
B. Binding of thaspine to the active site of topoisomerase IB

15. 1. The Plasmid relaxation assay demonstrate that thaspine is topoisomerase
IB inhibitor.
2. The Inhibition is reversible.
3. Thaspine inhibits the cleavage of DNA, this Inhibition is due to the inability of
enzyme to binds with DNA.
4. The inhibition mechanism of religation is same as that of Camptothecin.
5. Thus , finally thaspine act as both inhibitor and poison.

16. Prof. A.Desideri
Prof. Mattia Falconi
Paola Fiorani
Silvia Castelli
Oscar Vassalo
Cinzia Teausaro
Barbara Arno
Laura Zuccaro
Zeenat Jahan
Sara Viera
Aalborg University, Denmark : Erasmus Mundus Coordinator
&
European Commission .