p53

• Ability to prevent undue cell proliferation

• Plays a critical role in the host and tumor
response to radiation and chemotherapy
• It seems that p53 can be selectively
reactivated in tumor cells → target for gene
therapy
 MDM2
• Binds to the amino terminus of p53 and
blocks p53 activity
• Prediction: blocking p53-MDM2 interaction
would be a way to activate p53 without
having to expose cells to genotoxic drugs
In vivo activation of the p53 pathway by small-
molecule antagonists of MDM2

Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong

N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu E

Science (2004)303: 844-848

3 mechanisms to repress p53 by activation of MDM2-expression:

1. MDM2 binds p52 at transactivation domain and blocks its ability to

acitvate transcription
2. MDM2 is involved in the nuclear export of p53
3. MDM2 serves as a ubiquitin ligase that promotes p53 degradation
3 mechanisms to repress p53 by activation of MDM2-expression:

1. MDM2 binds p52 at transactivatin domain and blocks its ability to

acitvate transcription
2. MDM2 is involved in the nuclear export of p53
3. MDM2 serves as a ubiquitin ligase that promotes p53 degradation

Mdm2 gene is amplified or overexpressed in many human cancers

→ Activation of p53 pathway through inhibition of MDM2 has been

proposed as a novel therapeutic strategy
Series of cis-imidazoline
analogs that were named
Nutlins (for Nutley inhibitor)

Mode of binding of Nutlin in

MDM2-p53 complex
The treatment of cells with an inhibitor of MDM2-p53 binding should
result in
1. Stabilization and accumulation of p53
2. Activation of MDM2 expression
3. Activation of other p53-regulated genes and p53 pathway

At cellular level: cell cycle arrest in G1 and G2 phases and/or

apoptosis

Effect of MDM2 inhibitors on the cellular levels

of p53, MDM2 and p21

Mut p53 wt p53

Nutlin-1 treatment induces the
expression of p21 gene but not the p53
gene

Nutlins up-regulate p53 by means

of a posttranslational mechanism
Cell cycle arrest of wt p53 cancer cell lines
in G1 and G2 due to Nutlin-1
Effect of Nutlin-1 on growth and viability of
cultured cancer cells
 Effect of Nutlin-3a and Nutlin-3b on expression of p21, MDM2 and
p53 in wt p53 and mut p53-cells

→ Only Nutlin-3a activates expression of p21 and MDM2

Confirmation of the protein level by

Western blotting
Effect of Nutlin-3a and -3b on growth and viability of wt p53 and
mut53 cells
Ability of Nutlin-3 to induce apoptosis in cancer cells with
wt p53

After 48 hours Nutlin-3a incubation 45% of cell population showed

apoptosis
In vivo antitumor activity of Nutlin-3
 SUMMARY

™ Nutlins are a class of stuctures with high potency and

selectivity for reactivating p53 by inhibition of p53-MDM3
interaction
™ Activity of Nutlins is dependent on p53 status
™ There are two enantiomers of Nutlin-3. Activation of p53 and
apoptosis induction by Nutlin-3 is enantiomer specific
™ The data strengthen the notion that activation of p53 by
inhibition of MDM2 binding is a potentially valuable strategy
for treating cancer