Targeting Nucleic Acids with an Integrated Virtual and Actual Screen

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We seek a second renewal for our program to refine and apply an integrated virtual and actual screening platform for the discovery of new lead compounds to inhibit telomerase. Progress during the current funding period was outstanding, and we have achieved most of the specific aims proposed. During the next funding period, we will focus on the discovery of lead compounds that bind selectively to biologically important higher- order telomeric DNA quadruplexes and targeting the single strand telomere:POT1 interface. Telomerase has been identified as a target for over 20 years, but there are no approved drugs that inhibit its activity, so new approaches are required. We will inhibit POT1 (Protection of Telomere 1), a protein essential for telomerase activity, by targeting the telomeric DNA substrate, and by directly targeting the POT1 telomeric DNA binding function. We now propose fundamental studies that will continue to develop and refine our integrated screening platform. Nucleic acids remain underrepresented targets for small molecule therapeutic agents. There is mounting evidence to indicate that non-B DNA structures play prominent roles in gene expression and especially in the function of telomeres. Targeting these structural elements is an attractive and innovative strategy for the development of new therapeutic agents. The higher-order DNA quadruplex structures formed by the single strand of the human telomere remain a very poorly and improperly investigated target as traditional structural biology approaches have been unsuccessful. We have used a combination of computational modeling and experimental biophysics to derive consistent structural models for telomeric DNA sequences of up to 192 bases, or eight quadruplex repeats, in the current grant period. The higher-order quadruplex stacking interfaces thus represent a new target for small molecule stabilization of telomeric DNA. This stabilization should inhibit telomerase by locking the substrate DNA in an unusable form. An even more under represented target than non-B DNA is the nucleic acid-protein interaction, particularly single stranded DNA:protein complexes, as exist with POT1. We will target both the DNA substrate and the DNA-binding function of POT1, which could lead to synergistic inhibition of telomerase. This is a natural progression from our current funding period where we targeted biologically relevant quadruplex structures, to telomere-like higher-order quadruplex nucleic acid structures and the telomere ssDNA binding protein POT1, which is essential for telomeric DNA replication. The Specific Aims are: 1) Targeting higher-order telomeric quadruplex structures using integrated virtual and actual screens; 2) Characterizing the specific interactions of POT1 with higher-order quadruplexes; 3) Targeting POT1 using integrated virtual and actual screens; 4) Characterizing the biological properties of higher-order G-quadruplexes and POT1 binding agents. !