Molecular Design, Synthesis, and Pharmacology of Targeted Protein Degraders for the Checkpoint Kinase ATR

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About three billion base pairs are replicated within each mammalian cell cycle. Chemotherapeutics kill tumor cells through the induction of DNA replication stress and DNA damage. Exogenous and endogenous DNA stress activate checkpoint kinases, which slow down the cell cycle and initiate DNA repair. The apical checkpoint kinase ataxia telangiectasia-and-RAD3-related (ATR) is activated by stalled DNA replication forks and single strand DNA breaks. Preclinical and clinical studies have demonstrated the efficacy of ATP-competitive ATR inhibitors in combination with chemotherapeutics. Proteolysis-targeting-chimeras (PROTACs) are modern agents that inhibit and eliminate their target proteins by the ubiquitin-proteasome system. In preliminary work, we have synthesized and tested the first PROTAC for ATR in various cell systems. We demonstrate that the cereblon-targeting PROTAC Abd110 decreases ATR dependent on the E3 ubiquitin ligase cereblon and proteasomal activity. Abd110 synergistically induces apoptosis (programmed cell death) of acute myeloid and lymphatic leukemia cells when combined with the clinically used ribonucleotide reductase inhibitor hydroxyurea. We aim to optimize our PROTACs by structure-based design (using available X-ray structures of ATR and ternary PROTAC complexes of other kinases), in vitro testing, and cellular characterization. This includes pharmacological selectivity studies, targeted protein analyses, biochemical cell fractionation, flow cytometry, and genetic overexpression and knockout strategies. Promising candidates can be selected based on in vitro testing using recombinant ATR. We want to test and molecularly understand anti-leukemic effects of ATR PROTACs in combination with chemotherapeutics in a larger panel of leukemic cells and co-culture systems. This will involve analyses of DNA replication stress and DNA damage signaling. To comprehensively reveal the specificity and targets of ATR PROTACs, we will use global proteome, phospho-proteome, and transcriptome analyses. Furthermore, we will try to set up additional targeted proteolysis concepts, such as autophagy-targeting-chimeras (AUTOTACs) and chaperone-mediated protein degraders (CHAMPs/HEMTACs) for ATR. The degraders under study will be powerful tools to identify the downstream targets and biological functions of the catalytic activities and the structural/scaffolding properties of ATR. Moreover, such compounds could prospectively become new treatment options for difficult-to-treat leukemia.