PROJECT SUMMARY Highly recurrent TERT (the catalytic subunit of telomerase) promoter mutations in human familial and sporadic melanoma lead to a 2-4 fold increase in TERT transcription and telomerase activation, making telomerase an attractive target for melanoma cancer therapies. The development of effective small-molecule inhibitors of telomerase has been hindered by the lack of high-resolution structural data on telomerase. We used the TERT structure determined by X-ray crystallography to screen >500,000 compounds by in silico methods. This approach allowed us to identify a set of small molecules containing a similar scaffold that inhibited the enzymatic activity of telomerase. We have obtained the X-ray co-crystal structure of TERT bound to one of these compounds, revealing a novel and unexpected allosteric binding site, namely the FVYL pocket, located on the surface of the TERT thumb domain. This is the first-ever ligand-TERT co-crystal structure that has been solved to date. Using biochemical assays, we showed that the FVYL pocket binds telomerase RNA (TER), and therefore our initial lead acts by inhibiting the TERT – TER association and telomerase RNP assembly. We have also found compounds that can selectively inhibit the growth of telomerase-positive human melanoma cell lines, but show little growth inhibition of telomerase negative cancer and non-transformed cells in culture. The previous telomerase inhibitor BIBR-1532 was also examined. We determined the BIBR-1532- TERT co-crystal structure, showing that it binds to the same general area, but in a slightly different manner, as do the ligands that we identified by computational prescreening. We also found that BIBR-1532 had substantially less efficacy with a delayed action at killing melanoma cells than does the top hit from our own studies. The aims of this proposal are to (A) use structure-based design methods combined with medicinal chemistry to improve potency for the inhibition of telomerase, (B) evaluate the biochemical activity and specificity for telomerase while investigating the cellular pathways perturbed on telomerase inhibition, and (C) probe the in vivo melanoma oncolytic activity of the telomerase inhibitors. We are uniquely positioned to accomplish the work described because of our ability to iteratively obtain additional ligand-TERT co-crystal structures, and our expertise in the rapid parallel synthesis of new compound libraries, taking advantage of >20,000 reagents and starting materials available onsite (>800 boronic acids). Our research may provide a new approach for the structure-based design of small-molecule inhibitors of telomerase, and may reinvigorate research in the area of small-molecule telomerase inhibitors, eventually resulting in new drugs to treat melanoma alone or in combination with existing chemo- or immunotherapies. The research team is led by an expert in telomerase (Skordalakes) at The Wistar Institute, along with experienced pharma researchers in ...