A Cell-Based, High Throughput Screening Assay to Identify Small Molecule Compounds that Derepress Endogenous hTERT Expression Using Toxic hTR Template Mutants

Jesse Graham, Jessica Wheeler, Lancer K. Brown, Christopher A. Foster, Owen McGettrick, JianZhang,Jennifer Greeson,Rathnam Chaguturu, William H. Andrews, Daniel Hickman, Beril Karakas, Penelope Burke, Federico Gaeta, MieczyslawA. Piatyszek, Laura A. Briggs

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

www.sierrasci.com

Abstract

Human telomerase is the subject of intense research because of its critical function in cellular immortality via telomere length maintenance. Presence of telomerase activity is strongly associated with the immortality of embryonic stem and cancer cells. The human telomerase complex is composed of human Telomerase Reverse Transcriptase protein(hTERT) and human Telomerase RNA (hTR). Expression of telomerase activity is controlled mainly at the level of hTERT transcription. However,the specific mechanisms of repression or silencing of the hTERT promoter region remain unclear. Therefore, the purpose of our screen is to find small molecules which will relieve repression of the hTERT promoter.This should allow for transcription of hTERT mRNA and consequently the assembly of active telomerase. Two cell lines we reengineered by infecting human fetal lung fibroblasts(MRC5) with lentiviral vectors containing either a mutanth hTR (CTAGCG) or a wild type hTR (TTAGGG) template sequence.This mutant sequence was chosen because of its ability to reduce hTERT positive cell growth. Both viral constructs also express an siRNA sequence that inactivates the endogenous wildtype hTR.The hTR sequences contained within the lentiviruses are not affected by the siRNA sequence because they contain two additional base changes around the template region. When hTERT is expressed in mutant hTR cells dysfunctional CTAGCG telomeric repeats are presumably added at the ends of telomeres. The addition of mutated telomeric sequences are believed to cause the cells to become unhealthy and inhibit their growth. At the same time, the wild type hTR cells with induced telomerase activity proliferate as normal. Currently we have screened 49,524 compounds and have detected 618(1.25%) active compounds (primary hits).

SSLC Chemical Library

310,000 Diversified Chemical Compound Library

A pre-designed collection of 'drug-like' small molecules selected to meet the Lipinski's Rule of 5, and optimized to cover the broadest part of biologically relevantpharmacophorediversity space

2,000 Pharmacologically Characterized Chemical Compounds from MicroSource-

Constitutes a wide range of structural diversity-Potential to evaluate drugs and biochemical tools withknown biological profiles-Opportunity to explore the potential of novel structural and topographical diversity

Conclusions

A high throughput screening assay was developed to screen our compound libraries for small molecules inducing endogenous hTERTexpression in MRC5 cells.

Thus far 49,524 compounds have been screened. We identified 618 Active Compounds (1.25%) and 3364 CytotoxicCompounds (6.79%).

Cytotoxiccompounds will be "cherry picked" and serial diluted by a third from 33.3 µM to 11.1 µM to 3.7 µM to 1.23 µM. The dilute toxic compound plates will then be screened again.