Development of small-molecule autophagy modulators

Division Of Basic Sciences - Nci

Investigators

Abstract

Autophagy has been implicated in numerous diseases, including many cancers, but the difficulty of selectively targeting this pathway has presented major challenges for therapeutic development. My laboratory aims to discover novel autophagy modulators to better understand the fundamental roles of this pathway in human biology and disease. Inhibition of kinases involved in the early stages of the pathway and inhibition of lysosome functions in the late stages affect other cellular processes in addition to autophagy, because these protein and lipid kinases and lysosomes have other roles in the cell. Our approach to this challenge involves two complementary strategies: (1) targeting key protein-protein interactions that are uniquely involved in autophagy initiation to selectively inhibit autophagy and (2) applying a phenotypic approach combined with detailed target identification studies to discover autophagy modulators with new molecular targets and mechanisms. The development of selective, small-molecule autophagy modulators with defined mechanisms will enable detailed investigation of this important homeostasis pathway in fundamental biological processes and as a targetable pathway for therapeutic intervention. (1) Development of selective autophagy inhibitors that target protein-protein interactions to impact cancer therapy. Autophagy contributes to tumor progression by alleviating cellular stress and providing nutrients for the increased metabolic demands of rapidly dividing cells while facilitating the establishment of a metastatic niche. Our work to develop autophagy-selective compounds will enable evaluation of the role of autophagy in multiple types of cancer, with an initial focus on ovarian cancer, to assess the therapeutic potential of targeting autophagy as a frontline therapy and as a strategy to overcome resistance and recurrence. Our group has developed high-throughput assays to identify novel small-molecule inhibitors of key protein-protein interactions involved in the initiation of autophagy and autophagosome formation, including a bioluminescent resonance energy transfer (BRET) assay for the Beclin1-ATG14L interaction and a fluorescence polarization assay for the ATG5-ATG16L1 interaction. Our newly discovered inhibitor of the Beclin1-ATG14L interaction (Compound 19) inhibits the formation of the lipid kinase VPS34 Complex I that initiates autophagy. VPS34 is also required for endosomal trafficking through formation of a separate complex, Complex II. Inhibitors of VPS34 kinase activity inhibit both Complex I and Complex II formation, thereby inhibiting both autophagy and trafficking, which has limited clinical applications due to off-target effects. By selectively disrupting Complex I, our Compound 19 inhibits autophagy without affecting endosomal trafficking. We synthesized 83 small-molecule analogues and evaluated structure-activity relationships to reveal key regions, moieties, and functional groups that impact the potency and physicochemical properties of Compound 19 and impart selectivity for VPS34 Complex I over Complex II (J. Med. Chem. 2025, 68, 1645-1667). Through these efforts, we also identified an analogue with improved solubility and potency compared to Compound 19. The information obtained from these studies will facilitate further optimization of Compound 19 to enable evaluation of selective autophagy inhibition as a therapeutic strategy in cancer. The ATG5-ATG16L1 interaction is required for conjugation of LC3-I to phosphatidylethanolamine to form LC3-II, which is incorporated into the autophagosome membrane to facilitate membrane elongation, closure, and cargo recruitment. Our fluorescence polarization assay for the ATG5-ATG16L1 interaction is amenable to compound multiplexing, and this capability enables efficient screening of large compound collections. We have further optimized this assay to enable evaluation of complex mixtures of natural products to support bioactivity-guided fractionation to identify compound classes with novel biological activities. (2) Phenotypic discovery of novel molecular targets that regulate autophagy to develop new therapeutic strategies to treat cancer, neurodegeneration, and aging. Maintenance of proteostasis is critical for healthy aging and brain health, and the autophagy pathway has an important role in this process. Compounds that enhance autophagic flux could potentially provide neuroprotective effects in diseases that are characterized by protein misfolding, aggregation, and/or accumulation and resultant cytotoxicity. A major strength of phenotypic assays is the ability to rapidly identify compounds with the desired effect in cells, and when combined with subsequent target identification studies, new molecular targets for therapeutic development can be discovered. Using a phenotypic screening approach to identify small molecules that modulate the autophagy pathway, we recently discovered an autophagy modulator that improves disease-relevant phenotypes in cells from patients with a rare genetic disease. Our initial mechanistic studies indicate that this autophagy modulator may be serving as a small-molecule chaperone that prevents mutant protein degradation, reduces cellular stress, and potentially promotes folding and/or trafficking of misfolded proteins to restore protein function (manuscript in preparation). We have also identified an autophagy activator using this phenotypic approach and completed initial optimization efforts to provide RH1115. We previously identified lamin A/C and LAMP1 as targets of RH1115, and we are currently studying the mechanism of how binding of RH1115 to these proteins impacts their function and localization and how this modulates autophagy. Through iterative synthesis of an additional 43 analogues, we have been able to further optimize RH1115 and address metabolic liabilities to generate compounds with improved potency, reduced cytotoxicity, and improved microsomal stability (manuscript in preparation) to enable in vivo efficacy studies in disease-relevant models.