Book of abstract 2008

Avoiding systemic toxicity of the TNF superfamily ligands: induction of
cell death without ligands
Vanja Smilović1, Apolonija Bedina Zavec1, Radovan Komel1, Vladka Gaberc Porekar1,
Viktor Menart1 ,2
1National Institute of Chemistry, Ljubljana, Slovenia; 2Lek Pharmaceuticals, Ljubljana, Slovenia
Tumor necrosis factor alpha (TNF-α) is a cytokine capable of inducing hemorrhagic
necrosis and sometimes even full regression of tumors in vivo. Unfortunately, systemic
administration of TNF-α as an anti-cancer drug is not possible because of its severe adverse
effects. Lately, TNF-α analogues with potent antitumor activity and much lower systemic
toxicity have arisen, diminishing, but not completely eliminating adverse effects of TNF-α
therapy. Two other members of TNF superfamily, FAS ligand and TRAIL have also been
extensively investigated over the last two decades.
Signal transduction begins with ligand binding to the receptor resulting in clustering of
receptors on the extracellular side of the membrane. A cluster of at least two receptors is
required for induction of a conformational change in the intracellular part of the receptor
triggering the activation of the downstream cascade. To avoid ligand toxicity an interesting
alternative approach would be anti-cancer therapy without ligands.
In order to achieve clustering of the receptors without ligands, we introduced histidine
tags (HIS-10) into the extracellular part of the TNF-R1 molecule. Interactions of
histidines with transition metal ions in biological systems have been known for a long time
and they have also been successfully employed for purification of histidine rich proteins
in Immobilized Metal Affinity Chromatography (IMAC). In our case, HIS-10 tags in
the extracelullar parts of TNF-R1 receptors could serve for association via Zn 2+ ions and
biologically compatible chelating molecules, such as phytic acid and TETA (1,4,8,11-
Tetraazaciclotetradecane-1,4,8,11-acetic acid). This could lead to clustering of receptors
and consequently
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to activation of the downstream cascade.
HIS-10 tag has also been introduced into the truncated TRAIL-R1 molecule (in which
the extracellular part of the TRAIL-R1 molecule was deleted) to prove that clustering of
receptors is enough for inducing the downstream cascade and the extracellular parts of
TRAIL-R1 receptors are not required for signal tranduction. This approach would be
applicable for the whole superfamily of TNF receptors and also other situations where
signaling depends on protein clustering.
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