Development Of An Hts Assay To Screen For Inhibitors Of Nbs1-Atm Interactions

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO Developing novel sensitizers for cancer radiotherapy has been accelerated by recent advances of our understanding on how human cells respond to DNA damaging agents. One central element in determining the cellular response to irradiation (IR) is the ATM kinase, mutation of which causes the human autosomal recessive disorder ataxia-telangiectasia (A-T). A-T is characterized by progressive neuro-degeneration, a variable immunodeficiency, an extremely high predisposition to the development of lymphoid malignancies and hypersensitivity to radiation. Because ATM is central to cellular responses to IR, blocking its activity could make tumors much more sensitive to radiation. Although specific ATM inhibitors have shown promising preliminary results, several obstacles of applying these methods in the clinical setting exist. For example, due to the pleiotropic effects of the mutation, such as neuro-degeneration, immunodeficiency and cancer predisposition, the outcome of directly inhibiting ATM kinase activity can be complicated, as it is unclear whether the only effect of these reagents will be to confer radiosensitization. Instead of directly inhibiting ATM kinase activity to increase radiosensitivity, an alternative approach is to target IR-induced ATM activation, as this may directly lead to an increase in radiosensitivity without interfering with other important functions of ATM. Activation of ATM in response to IR requires interaction with the extreme C-terminus of NBS1. We have recently made proof-of-principle discoveries showing that targeting NBS1-ATM interactions confers radiosensitization. In order to identify small molecules that can specifically inhibit NBS1-ATM interactions, we have developed a fluorescence polarization (FP) assay feasible for high throughput screening (HTS). The in vitro binding assay is carried out using Texas Red-labeled NBS1 small peptides with GST-conjugated ATM Heat-Repeat domains. Several Texas Red-labeled peptides containing the wild-type or mutant NBS1 C-terminal sequences were synthesized. For the GST ATM production, we subcloned ATM Heat-repeat domain cDNA into a pGEX-2T bacterial expression vector and purified GST-ATM protein. The dynamic range of the assay is defined as the polarization difference between the bound peptide and the free peptide. The stability of the FP assay to variables such as dose responses, incubation time, total fluorescence, and dimethyl sulfoxide (DMSO) tolerance was assessed. The assay tolerates 5% of DMSO without significant loss of signals, which allows for testing of wide ranges of compound concentrations and enables screening poorly soluble compounds. Under optimized conditions, a Z\#8242; factor of 0.71 was achieved in a 384-well format for high throughput screening, indicating a feasible and robust assay for HTS. This assay has been enrolled in the NIH Roadmap Initiative Molecular Libraries Program for large scale HTS. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2303.