1. Characterization of the effect of Cyclometalated Gold (III)
on the human topoisomerase IB catalytic cycle.
Supervisor By…
Prof. Alessandro Desideri Prafulla Katkar

2. Human Topoisomerase IB :
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.
Topoisomerase Inhibitors : 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)
topothecan
2. Topo Inhibitors : Inhibit cleavage of DNA
Cell Death (Apoptosis)

3. Mechanism of Action :
Topoisomerase IB Topo poison

4. Gold (III) Compound :
[Au(C^N^C)(IMe)]CF3SO3
Gold(III) complex modified by dianionic [C^N^C]2- and
neutral auxiliary N-heterocyclic carbene (NHC) ligands.

5. Gold as a Drug :
Gold (III) complex is a Square planner d8 metal complex that have been
known to exhibit promising anticancer properties. (K.S.Lovejoy et al 2009)
1. by covalent crosslink of d8 metal ions to DNA,
2. by intercalation of the planar metal complexes between the DNA
base pairs.
3. and/or by inhibition of enzyme activities.
Aims of the work :
1. Investigation of Mechanism of effect of Gold III on Topoisomerase
IB whether it is Topo poison or inhibitor.
2. Analyzing its effect on different steps of enzyme catalysis.

6. Our Work...
Plasmid relaxation assay :
Effect of Gold (III) on topoisomerase IB relaxation :
Gold (III) inhibits the human topoisomerase IB
in a dose dependent manner
The inhibitory effect is irreversible

7. Effects of Gold (III) on cleavage/religation equilibrium :
Time course :
The duplex is incubated at 37°C for 5’,15’ and 30’
with the enzyme in presence of DMSO (lane 2-4)
as solvent inhibitory activity control, or in
presence of 50μM Gold(III) (lane 3-6), 50μM CPT
(lane 8-10), and 50μM Gold(III) 5’ Preincubation
at 37°C with subsequent addiction of 50μM CPT
(lane 11-13).
religated oligo
trapped cleavable complex
Either Gold (III) inhibits the cleavage or induces a faster religation,
so that the cleavable complex cannot be formed or cannot be stabilized by CPT.

8. Analysis of the religation rate :
Gel analysis of the religation kinetics for topoisomerase IB in absence
(lanes 2–10) or presence of 50uM Gold(III) (lanes 11–19)
GOLD
DMSO
DMSO
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
PRE-GOLD
Percentage of the religation products
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
plotted at different times

9. Analysis of the cleavage rate :
In the presence of 50μM Gold (III) the cleavage
DMSO reaction is clearly inhibited.
The band corresponding to the cleaved substrate is
% Cleavage
quite weak and not more than 30% of the product
can be obtained even after one hour incubation.
GOLD
Cleavage inhibition is almost complete when
PRE-GOLD
the enzyme is pre-incubated with 50μM Gold (III)
time min

10. EMSA : Electrophoretic Mobility Shift Assay
C DM G PG Cpt C DM G PG Cpt
Top1–DNA complex
C – Control
DM – DMSO
G – Gold
PG – Preincubated Gold
Cpt – Camptothecin DNA Substrate
Gold III binds with the topo IB, thus inhibiting the Topo – DNA complex

11. 1. The Plasmid relaxation assay demonstrate that Gold III is topoisomerase
IB inhibitor.
2. The Inhibition is irreversible.
3. The secondary visible effect of Gold (III) is inhibition of topoisomerase I
cleavage step. This Inhibition is due to the inability of enzyme to binds with
DNA.
4. The inhibition mechanism is different than Camptothecin.
5. Gold (III) have no effects on the religation step.
6. EMSA demonstrate that Gold (III) binds with topoisomerase, thus preventing
Top1–DNA complex formation.

12. Prof. A.Desideri
Prof. Raymond Wai-Yin sun
Prof. Mattia Falconi
Paola Fiorani
Silvia Castelli
Oscar Vassalo
Cinzia Teausaro
Barbara Arno
Laura Zuccaro
Zeenat Jahan
Aalborg University, Denmark : Erasmus Mundus Coordinator
&
European Commission .