Characterization of the effect of Cyclometalated Gold (III) on the human topoisomerase IB catalytic cycle.

Supervisor By…Supervisor By… Prof. Alessandro DesideriProf. Alessandro Desideri Prafulla Katkar Prafulla Katkar

Human Topoisomerase IB :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 Topoisomerase Inhibitors : Most anticancer drugs have their topoisomerase as their molecular target.

1.Topo poisons : Stabilizes Topo-DNA cleavable complex and inhibit religation. (Camptothecin Camptothecin and its derivatives topothecantopothecan, GematecanGematecan approved in clinical trials)

2. Topo Inhibitors : Inhibit cleavage of DNA

Cell Death (Apoptosis)

Mechanism of Action :Mechanism of Action :

Topoisomerase Topoisomerase IB IB Topo poison Topo poison

Gold(III) complex modified by dianionic [C^N^C]dianionic [C^N^C]2- 2- andneutral auxiliary N-heterocyclic carbene (NHC) carbene (NHC) ligands.

Gold (III) Compound :Gold (III) Compound :

[Au(C^N^C)(IMe)]CF3SO3

Gold as a Drug :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 :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.

Our Work...Our Work...

Effect of Gold (III) on topoisomerase IB relaxation :Effect of Gold (III) on topoisomerase IB relaxation :

The inhibitory effect is irreversibleThe inhibitory effect is irreversible

Plasmid relaxation assay :Plasmid relaxation assay :

Gold (III) inhibits the human topoisomerase IBGold (III) inhibits the human topoisomerase IB in a dose dependent manner in a dose dependent manner

Effects of Effects of Gold (III)Gold (III) on cleavage/religation equilibrium : on cleavage/religation equilibrium :

Time course :Time course :

trapped cleavable complextrapped cleavable complex

religated oligoreligated oligo

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).

Either Gold (III) inhibits the cleavage or induces a faster religation, 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.so that the cleavable complex cannot be formed or cannot be stabilized by CPT.

Analysis of the religation rate :Analysis of the religation rate :

Gel analysis of the religation kinetics for topoisomerase IB in absenceGel analysis of the religation kinetics for topoisomerase IB in absence (lanes 2–10) or presence of 50uM Gold(III) (lanes 11–19) (lanes 2–10) or presence of 50uM Gold(III) (lanes 11–19)

Percentage of the religation products Percentage of the religation products plotted at different timesplotted at different times

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

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

DMSODMSO

GOLDGOLD

PRE-GOLDPRE-GOLD

DMSODMSO

Analysis of the cleavage rate :Analysis of the cleavage rate :

time mintime min

% C

leav

age

% C

leav

age

In the presence of 50μM Gold (III) the cleavage reaction is clearly inhibited.

The band corresponding to the cleaved substrate isquite weak and not more than 30% of the productcan be obtained even after one hour incubation..

Cleavage inhibition is almost complete when the enzyme is pre-incubated with 50μM Gold (III)PRE-GOLDPRE-GOLD

DMSODMSO

GOLDGOLD

C DM G PG Cpt C DM G PG Cpt

C – ControlC – ControlDM – DMSODM – DMSOG – GoldG – GoldPG – Preincubated GoldPG – Preincubated GoldCpt Cpt – – CamptothecinCamptothecin

Top1–DNA complexTop1–DNA complex

DNA SubstrateDNA Substrate

EMSA : Electrophoretic Mobility Shift AssayEMSA : Electrophoretic Mobility Shift Assay

Gold III binds with the topo IB, thus inhibiting the Topo – DNA complexGold III binds with the topo IB, thus inhibiting the Topo – DNA complex

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.

6. EMSA demonstrate that Gold (III) binds with topoisomerase, thus preventing Top1–DNA complex formation.

5. Gold (III) have no effects on the religation step.

Prof. A.DesideriProf. Raymond Wai-Yin sunProf. Mattia Falconi Paola FioraniSilvia CastelliOscar VassaloCinzia TeausaroBarbara ArnoLaura ZuccaroZeenat Jahan

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