A Cell-Based, High Throughput Screening Assay to Identify Small Molecule Compounds that Derepress the Telomerase Minimal Promoter in a Transient Luciferase Expression System

Jian Zhang, Beril Karakas, Lancer K. Brown, Christopher A. Foster, Jesse Graham, Jennifer Greeson, William H. Andrews, Shawna Tanglao, Thomas Lovell, Hamid Mohammadpour, Federico Gaeta, Mieczyslaw Piatyszek, Laura A. Briggs, Rathnam Chaguturu

Sierra Sciences LLC, 250 S. Rock Blvd., Suite 130 Reno, NV 89502

www.sierrasci.com

Abstract

The presence of telomerase activity in human cells is strongly correlated to the expression of hTERT which is repressed in normal adult human cells. We are undertaking a screen for compounds that activate telomerase expression using a transient expression system similar to the one previously described by Won et al. (PNAS, 2004, vol. 101, p.11328-11333). The telomerase minimal promoter sequence was inserted into a promoter-reporter plasmid to drive expression of the luciferase coding sequence. The plasmid was then transfected into normal MRC5 cells (human fetal lung fibroblasts) which represses the promoter and gives background levels of luciferase activity. These transfected cells were used in a high throughput screening system to identify small molecules that derepress the telomerase minimal promoter turning on luciferase expression. During the screening process the MRC5 cells were transfected in bulk, seeded into 96-well plates and then the compounds are added to a final concentration of 33 uM for 48 hr treatment. Potential active compounds are identified by increased luciferase activity. We have presently screened 123,840 compounds and indentified 929 compounds that stimulate luciferase activity 3 to 20 fold over the DMSO negative controls. These 929 compounds represent several distinct chemical families. At least 7 of these active compounds have been shown to induce hTERT mRNA expression (RT-PCR) and telomerase activity (TRAP) in normal cells. Studies of these compounds with regards to specificity, selectivity, and an ability to extend cellular lifespan are ongoing.

SSLLC Diversivied Chemical Library

320,000 Compounds

• A pre designed collection of 'drug-like' small molecules selected to meet the Lipinski Rules, and optimized to cover the broadest part of biologically relevant pharmacophore diversity space


• Constitutes a wide range of structural diversity


• Potential to evaluate drugs and biochemical tools with known biological profiles


• Opportunity to explore the potential of novel structural and topographical diversity

Summary

Cell-based high throughput screen has been discovered to identify potential actives that derepress the hTERT minimal promoter in MRC5 cells

132,800 compounds were screened yielding 1,040 actives (300% above DMSO control) with a hit rate of 0.78%

So far several compounds have been positive for endogenous hTERT mRNA detected by RT-PCR

the replicative lifespan of cells treated with active compounds are currently under investigation

Path Forward

Continue Screening

Validation and hit to lead assessment

Expand structure activity relationships (SAR) of most promising active compounds

Confirm activity in additional cell lines

RT-PCR studies for hTERT mRNA levels

TRAP for hTERT enzymatic activity

Expand HTS effort to identify other active pharmacophores

Develop pharmacological models to evaluate activity in vivo

Expansion of lifespan?