Identification and Characterization of Novel Small-Molecule Inhibitors of the Replication Checkpoint.
Authors: Bradner James , Kim Yong-Son , Koehler Angela , Kawasumi Masaoki , Li Xiaodong , Schreiber Stuart L. , Nghiem Paul 
Published: October 2019

Background The replication (G2/M) checkpoint is principally mediated by the serine/threonine protein kinase ATR (ataxia telangiectasia mutated and Rad3-related). ATR is a large (350 kD) member of the phosphatidylinositol kinase related kinase family. After exposure to genotoxic or replication stress, ATR halts cell cycle progression, allowing DNA repair complexes time enough to restore the fidelity of the genome prior to cell division. Previous experiments have demonstrated that cancer cells with p53 mutation are critically dependent on ATR-mediated arrest of the cell cycle. Industrial approaches to identify ATR inhibitors have failed likely as a result of protein insolubility.

Methods We have undertaken a novel chemical genetic approach employing small molecule microarrays (SMMs) to identify molecules with high binding specificity for ATR. Three diversity-oriented combinatorial chemical libraries of more than 15,000 entities were generated by split-pool synthesis in solid phase on polystyrene macrobead supports. Compounds were robotically printed in microarray format on glass slides. Four analogs of FK506 were printed as positive controls. Extracts were prepared from mammalian cells transfected with over-expression constructs of FLAG-tagged ATR, FKBP12 and GFP. A protocol was developed and optimized for screening employing a primary anti-FLAG mouse monoclonal antibody and Cy5-fluorophore labeled anti-mouse antibody. Data analysis for small molecule binders was performed with GenePix software on an Axon Scanner. Biological activity of these molecules was analyzed in the context of mitotic spread and chromosomal fragility assays.

Results Protein expression and antibody fidelity was verified by Western blot. The lysate-based SMM screening approach was optimized and validated by recognition of an interaction between over-expressed, epitope-tagged FKBP12 and analogs of FK506. Six small molecule hits suggesting ATR binding were identified and verified by triplicate microarray assays. Positive compounds were structurally similar members of a dihydropyrancarboxamide library suggesting recognition of a common target. Mitotic spread analysis of cells treated with two of these molecules and hydroxyurea demonstrated the premature chromatin condensation phenotype characteristic of replication checkpoint inhibition. Chromosomal fragility was notably augmented by these molecules as well. Chemosensitivity following replication stress was witnessed in p53-negative cells relative to an otherwise identical wild-type cell line.

Conclusions Classical approaches to drug discovery are often limited by challenges in protein biochemistry such as protein size, solubility, activity and yield. We present compelling data that the small molecule microarray format can effectively be tailored for use with cellular lysates over-expressing a protein target of biological interest. Furthermore, we have used an optimized protocol to identify two novel, active small molecule inhibitors of the replication checkpoint (SMIRC-1 and SMIRC-2). The enhanced chemosensitivity in p53-negative cell lines supports a plausible role for ATR inhibitors as potentially useful chemotherapeutic agents.