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

Topoisomerase inhibtion by thaspine

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Page 1: Topoisomerase inhibtion by thaspine

A Natural Anticancer Agent Thaspine Targets Human Topoisomerase IB

Supervisor By…Prof. Alessandro Desideri Prafulla Katkar

Page 2: Topoisomerase inhibtion by thaspine

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.

Page 3: Topoisomerase inhibtion by thaspine

Most anticancer drugs have their topoisomerase as their molecular target.

Cell Death (Apoptosis)

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

Page 4: Topoisomerase inhibtion by thaspine

Topoisomerase IB Topo poison

Page 5: Topoisomerase inhibtion by thaspine

An alkaloid present in the cortex of south American tree Croton lechleriBeing 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.

Dragon’s Blood (Sangre di grado)

Page 6: Topoisomerase inhibtion by thaspine

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.

Page 7: Topoisomerase inhibtion by thaspine

Effect of Thaspine on topoisomerase IB relaxation :

Thaspine inhibits the human topoisomerase IBin a dose dependent manner

1 2 3 4 5 6 7

C 0 5 10 15 20 50 µM

Thaspine

NC

Relaxed

SC

Page 8: Topoisomerase inhibtion by thaspine

DMSO Thaspine Thaspine+E

Pre-incubation

NC

1 2 3 4 5 6 7 8 9 10 11 12 13

C 1 7.5 15 30 1 7.5 15 30 1 7.5 15 30 Time (Min)

SC

Relaxed

Pre-incubation of drug increase the inhibitory effect for short time

Then decreases gradually

Page 9: Topoisomerase inhibtion by thaspine

1 2 3 4 5 6 7 8 9

C 1:1 1:2 1:4 1:8 1:1 1:2 1:4 1:8DMSO Thaspine+E

Pre-incubation

NC

Relaxed

SC

The inhibitory effect of thaspine is reversible, as the effect is lost as the

dilution increases

Page 10: Topoisomerase inhibtion by thaspine

1 2 3 4 5 6 7 8 9 10 11 12 13

C 5 15 30 5 15 30 5 15 30 5 15 30

DMSO Thaspine CPT Taspine+E

Pre-incubation

Either thaspine inhibits the cleavage or induces a faster religation,

so that the cleavable complex cannot be formed.

Religated oligo

Cleavable Complex

Page 11: Topoisomerase inhibtion by thaspine

1 2 3 4 5 6 7 8 9 10 11 1213 14 15

C 0 0.5 1 5 10 20 30 0 0.5 1 5 10 20 30

DMSO Thaspine

----- Tasp

DMSO

The religation rate is slowed down in presence of thaspine as compared with DMSO

Page 12: Topoisomerase inhibtion by thaspine

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

C 0.5 1 5 10 20 60 0.5 1 5 10 20 60 0.5 1 5 10 20 60

DMSO Thaspine Thaspine+E

Pre-incubation

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.

Page 13: Topoisomerase inhibtion by thaspine

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

Page 14: Topoisomerase inhibtion by thaspine

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

Page 15: Topoisomerase inhibtion by thaspine

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.

Page 16: Topoisomerase inhibtion by thaspine

Prof. A.DesideriProf. Mattia FalconiPaola FioraniSilvia CastelliOscar VassaloCinzia TeausaroBarbara ArnoLaura ZuccaroZeenat JahanSara Viera

Aalborg University, Denmark : Erasmus Mundus Coordinator&European Commission .