ABSTRACT Autophagy is a major catabolic recycling process that is critical for cellular homeostasis, especially under stressful conditions, such as hypoxia and nutrient deprivation. Autophagy has been implicated in numerous disease states, including cancer. Cancer cells can utilize autophagy for proliferation and tumorigenesis, providing a means for survival in stressful conditions such as chemotherapeutic treatment. Therefore, autophagy inhibition shows promise as a treatment option in these cases. While current efforts have evaluated inhibition of the lipid kinase VPS34, the involvement of this kinase in both the autophagy (Complex I) and vesicle trafficking (Complex II) pathways leads to inhibition of both pathways. This lack of selectivity has led to the limited use of autophagy inhibition as a therapeutic strategy in cancer. Unlike VPS34, ATG14L is only found in the autophagy initiation complex, where it facilitates key protein-protein interactions (PPIs). The hypothesis of this proposal is that small- molecule inhibition of the ATG14L-Beclin 1 PPI will selectively disrupt Complex I and inhibit autophagy without disrupting other processes controlled by Complex II. To test this hypothesis, optimization of potency and in vitro DMPK of small molecule leads will be performed as described in Aim 1. To accomplish this, a cellular NanoBRET assay was optimized to develop a high-throughput screen (HTS) to discover molecules that inhibit the ATG14L- Beclin 1 PPI. An initial screen of 2560 compounds in this assay revealed 19 hits which inhibited the interaction. Of these 19 hits, one compound was selected due its dose-dependent inhibition of this PPI and its potency. Counterscreens revealed that this compound specifically inhibited Complex I formation and did not affect Complex II vesicle trafficking. A synthetic route to access analogues has been developed. The hit compound will be optimized for potency, toxicity, aqueous solubility, and microsomal stability to generate a lead with desirable properties for future in vivo studies. This lead molecule will be evaluated as an autophagy-specific probe through testing in Aim 2. Screening in 15 cancer cell lines will analyze single-agent efficacy and will identify biomarkers that predict sensitivity or resistance to autophagy inhibition. Chemotherapeutics will also be combined with the lead to determine if selective autophagy inhibition has additive or synergistic effects with established chemotherapeutics in resistant cell lines. The results of these studies will reveal the utility of autophagy inhibition as a therapeutic strategy in cancer and will enable selection of a patient population that could benefit from autophagy inhibition therapy. Collaborative efforts from the sponsor, co-sponsor, and their respective trainees will allow for feedback that improves experimental design, oral and written communication, and future direction of the work. Coupling this with the extensive Research Resource Center and ...