Page 68 - Biennial Report 2018-20 Jun 2021

experiments. Based on these experiments, a single residue was indicated to be essential for
NME2-telomerase interaction. Several 10-15mer peptides were designed based on this. Each of
these indicated some effect on reducing telomerase activity. Therefore, in the first aim the study
was initiated by screening the efficacy of telomerase targeting peptides in suppressing
telomerase activity. Out of four peptides of varying length, i.e. 10mer-Peptide 1; 12mer-Peptide
2; 14mer-Peptide 3 and 15mer- Peptide 4; Peptide 3 and 4 were narrowed down that showed
better repression of telomerase activity in the preliminary experiments. The effect of Peptides 3
and 4 was started to be tested along with scrambled 14-mer peptide, carrying the same amino
acid composition as peptide 3 using purified telomerase (hTERT+hTERc complex). This functional
telomerase complex was purified from HEK293T cells by over-expressing FLAG-tagged-
hTERT+hTERc complex, using anti-FLAG antibody. Thereafter, each of the above designed NME2
derived peptides was checked to see the effect of test peptides on telomerase activity. Both
peptide 3 and peptide 4 showed 0.4- and 0.5-fold repression, while the scrambled peptide
showed a 0.1-fold decrease in telomerase activity respectively. From these results it was inferred
that NME2 derived peptides 3 and 4 were both capable of repressing telomerase activity up-to
50%.

Next, the effect of peptides 3 and 4 was tested along with scrambled control on cell lysates from
fibrosarcoma cell line HT1080, breast cancer cell line MDA-MB-231 as sources of cellular
telomerase. In-vitro incubation of test peptides was done with respective cell lysates as done for
the above experiment. In both HT1080 and MDA-MB-231 cell lines, both peptide 3 and 4 caused
more than 90% repression of telomerase activity, while at the same concentration scrambled
peptide also showed some non-specific repression of telomerase activity. Non-specific effect of
scrambled peptide needs to be further explored and investigated. This could be a cell line
dependent issue and therefore effects might be dependent on the cancer types used for the
study.
Based on the in-vitro results experiments were designed to test the effect of NME2 derived
peptides on fibroblast and breast cancer cell lines. Since the peptides could not penetrate the
nucleus without a carrier, the effect of target peptide was first tested with lipofectamine 2000.
Unfortunately, lipofectamine with the peptide demonstrated extensive cytotoxicity. Therefore,
an alternate lipid-based chemistry approach was attempted using liposomes for peptide
delivery. Using liposomes encapsulating peptides, HT1080 (fibrosarcoma) and MDAMB231
(breast cancer) cells were treated at 12hr intervals using 2, 4 and 8ug of target peptide 3 with
respective scrambled control. The findings showed that NME2 derived peptides were capable of
suppressing telomerase activity inside the cells as well. To attain 60% silencing in telomerase
activity the cells were treated with 4ug of peptide every 12 hours. This dose was tested and
found not to be cytotoxic for normal primary cells. While this data was promising, the cells here
were also treated with specific mutant peptides that carried mutation at a single specific residue
predicted to be important in interaction with telomerase, based on our preliminary protein-
protein docking experiments. Unfortunately, the mutant peptide did not lose the repressive
activity of NME2 peptide. This aspect needs further exploration and mutant screening
experiments to specifically determine if this residue individually is the most crucial for NME2-
hTERT interaction and telomerase suppression.

On a second arm of the study, in collaboration with Dr. Chakraborty, new telomerase targeting
molecules were designed. These molecules are TFA (tri-fluroacetic acid) derivatives, that bind G-
quadruplexes (TKC1,2). hTERT gene promoter is well studied for tandem G-quadruplexes present
within its core-promoter. These are functionally significant in telomerase regulation. Therefore,
67