DNATWO, Inc. was founded out of Caltech to develop small molecule drugs targeting a new cancer target, DNA2, to treat triple-negative breast cancer (TNBC). We have spent decades unraveling the role of the DNA2 ATP-motor driven nuclease activity in relieving replication stress at the replication fork and carrying out repair at forks collapsed to double strand breaks (DSBs). Our scientific premise is that DNA2 is the Achilles’ heel of cancer cell proliferation, and that we can target it using small molecule inhibitors. Because activated oncogenes and inactivated tumor suppressors lead to replication stress and double strand breaks (DSB), a subset of tumors is dependent on DNA2 to support their growth. Breast cancer (BC), with over 281,000 new cases and 44,000 deaths expected in 2021 in the USA, is the second most common cancer in women. BC is a heterogenous disease, with 10-15% of cases being classified as basal like, predominantly TNBC. TNBC has the worst prognosis of human BC subtypes and is more prevalent in young women under 40, particularly African American women. A factor in the poor outcome is the dearth of targeted therapies. Our focus on DNA2 as a target in TNBC is consistent with 15-20% of TNBC cases being deficient in the breast cancer susceptibility genes BRCA1/2 critically important in combating replication stress. We have demonstrated that DNA2 is important for genome stability and survival in TNBC cell lines. A therapeutic index for DNA2 inhibitors is likely due to the high intrinsic levels of replication stress in tumors with mutated oncogenes or tumor suppressors as compared to normal cells (i.e. sensitizing tumor cells), as well as the presence of alternative repair pathways present in normal cells (i.e. protecting normal cells from loss of DNA2 function). We have shown this experimentally by comparing potency of DNA2i in a normal/diploid cell model as well as cancer cell lines in vitro. We have previously described small molecule DNA2 inhibitors and demonstrated their biochemical selectivity and mode of action in vitro in breast cancer cells. This Phase I award will allow us to pursue two milestones: Hit-to-Lead development and the demonstration of in vivo efficacy. Aim 1 focuses on Hit-to-Lead optimization on the most advanced DNA2 inhibitor (DNA2i) scaffolds. The goal is to improve drug-like properties and DNA2 biochemical and cellular potency without loss of target specificity, resulting in lead molecules with nM potency in cells with minimum activity against other DNA repair enzymes, and promising in vitro ADME properties to support in vivo testing. Aim 2 will evaluate lead compounds in a mouse PK study to confirm in vivo exposure followed by an in vivo mouse TNBC xenograft study. The goal is a lead DNA2i molecule with demonstrated in vivo efficacy. Successful completion of this project will allow us to advance to lead optimization studies and further pharmacology, IND-enabling studies (GLP, PK, and toxicology) in Phase II....