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. !

Public Health Relevance

This project will refine and apply an integrated computational and experimental screening platform for the discovery of new drugs that target specific structures of functional importance within the genome. The current funding period for this project was successful and productive, and we will continue our efforts. Several novel telomeric functionally important quadruplex structures were characterized. We will characterize the interactions of these with their protein partner and use this information to target both.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077422-10
Application #
9690716
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter
Project Start
2007-02-01
Project End
2021-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
10
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Louisville
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
057588857
City
Louisville
State
KY
Country
United States
Zip Code
40292
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Del Villar-Guerra, Rafael; Gray, Robert D; Trent, John O et al. (2018) A rapid fluorescent indicator displacement assay and principal component/cluster data analysis for determination of ligand-nucleic acid structural selectivity. Nucleic Acids Res 46:e41
Del Villar-Guerra, Rafael; Trent, John O; Chaires, Jonathan B (2018) G-Quadruplex Secondary Structure Obtained from Circular Dichroism Spectroscopy. Angew Chem Int Ed Engl 57:7171-7175
Del Villar-Guerra, Rafael; Gray, Robert D; Chaires, Jonathan B (2017) Characterization of Quadruplex DNA Structure by Circular Dichroism. Curr Protoc Nucleic Acid Chem 68:17.8.1-17.8.16
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Bon?ina, MatjaĆŸ; Vesnaver, Gorazd; Chaires, Jonathan Brad et al. (2016) Unraveling the Thermodynamics of the Folding and Interconversion of Human Telomere G-Quadruplexes. Angew Chem Int Ed Engl 55:10340-4
Miller, M Clarke; Ohrenberg, Carl J; Kuttan, Ashani et al. (2015) Separation of Quadruplex Polymorphism in DNA Sequences by Reversed-Phase Chromatography. Curr Protoc Nucleic Acid Chem 61:17.7.1-18
Chaires, Jonathan B; Dean, William L; Le, Huy T et al. (2015) Hydrodynamic Models of G-Quadruplex Structures. Methods Enzymol 562:287-304
Chaires, Jonathan B (2015) A small molecule--DNA binding landscape. Biopolymers 103:473-9
Le, Huy T; Dean, William L; Buscaglia, Robert et al. (2014) An investigation of G-quadruplex structural polymorphism in the human telomere using a combined approach of hydrodynamic bead modeling and molecular dynamics simulation. J Phys Chem B 118:5390-405

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