Abstract 2907: A first-in-class series of potent and selective macrocyclic ATR inhibitors

Abstract ATR, a phosphatidyl-inositol kinase-related kinase (PIKK), is a promising target for cancer treatment. Although ATR inhibitor (ATRi)-based therapies show increased activity in tumors that harbor various cancer-associated alterations, the therapeutic index of this drug class has remained a challenge. One potential risk factor in causing drug-related adverse is limited selectivity for the drug target (i.e. off-targeting). In small molecule drugs, macrocyclic structures can reduce the number of conformations possible and, thus, promote selectivity for their targets. Therefore, to increase the selectivity of ATRi and potentially reduce adverse effects of treatment, Aprea Therapeutics developed a first-in-class series of macrocyclic ATRi to potentially increase the tolerability of treatment while maintaining biomarker-driven efficacy. These macrocyclic ATRi are highly potent, with IC50s below 20 nM based on in vitro biochemical kinase assays and cellular inhibition of phosphorylation on S345 of CHK1, a direct ATR substrate. Notably, members of the Aprea series of macrocyclic ATRi demonstrate minimally off-target other PIKKs both in in vitro kinase assays and in cultured cells, with off-targeting of other PIKKs (ATM, PRKDC, MTOR, and SMG1) occurring at concentrations that are hundreds of fold greater than that required to inhibit ATR. Cell proliferation and replication fork stability assays confirm low nanomolar EC50s as single agents at levels that correlate with their ability to inhibit ATR. As expected based on ATR’s role in the replication stress and DNA damage responses, the antiproliferative effects of members of this macrocyclic ATRi series were amplified by co-treatment with DNA-damaging chemotherapies, such as topoisomerase inhibitors, PARP inhibitors, and platinum agents. Mouse xenograft tumor studies with one of the macrocyclic series members, ATRN-119, indicate single-agent activity in colon and prostate cancer models. Moreover, the growth of orthotopically transplanted BRCA2-deficient ovarian cancer PDX tumors is significantly impeded by ATRN-119 single agent treatment, and the combination of ATRN-119 with PARP inhibitor treatment in this model synergistically suppresses tumor growth and causes tumor regression. Overall, Aprea’s new class of macrocyclic ATRi represents a new class of ATRi with selectivity for inhibiting ATR over other PIKKs, has cellular activities that match biochemical and cellular inhibition of ATR, and exhibits tumor suppressive effects and a single agent and in combination with PARP inhibition. ATRN-119 is now progressing through Phase I clinical dose escalation studies. Citation Format: Molly M. Hansbarger, Stephen J. Rocca, Tina Gill, Hank Breslin, Lanqi Jia, Erin M. George, Inna Rom, Teresa Lee, Jonathan Weinstein, Fiona Simpkins, Eric J. Brown, Oren Gilad. A first-in-class series of potent and selective macrocyclic ATR inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2907.