PHY34 inhibits autophagy through V-ATPase V0A2 subunit inhibition and CAS/CSE1L nuclear cargo trafficking in high grade serous ovarian cancer.

PHY34 is a synthetic small molecule, inspired by a compound naturally occurring in tropical plants of the Phyllanthus genus. PHY34 was developed to have potent in vitro and in vivo anticancer activity against high grade serous ovarian cancer (HGSOC) cells. Mechanistically, PHY34 induced apoptosis in ovarian cancer cells by late-stage autophagy inhibition. Furthermore, PHY34 significantly reduced tumor burden in a xenograft model of ovarian cancer. In order to identify its molecular target/s, we undertook an unbiased approach utilizing mass spectrometry-based chemoproteomics. Protein targets from the nucleocytoplasmic transport pathway were identified from the pulldown assay with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing a likely candidate protein. A tumor microarray confirmed data from mRNA expression data in public databases that CAS expression was elevated in HGSOC and correlated with worse clinical outcomes. Overexpression of CAS reduced PHY34 induced apoptosis in ovarian cancer cells based on PARP cleavage and Annexin V staining. Compounds with a diphyllin structure similar to PHY34 have been shown to inhibit the ATP6V0A2 subunit of V(vacuolar)-ATPase. Therefore, ATP6V0A2 wild-type and ATP6V0A2 V823 mutant cell lines were tested with PHY34, and it was able to induce cell death in the wild-type at 246 pM while the mutant cells were resistant up to 55.46 nM. Overall, our data demonstrate that PHY34 is a promising small molecule for cancer therapy that targets the ATP6V0A2 subunit to induce autophagy inhibition while interacting with CAS and altering nuclear localization of proteins.

Systematic Diversity-Oriented Synthesis of Reduced Flavones from γ-Pyrones to Probe Biological Performance Diversity.

Diversity-oriented synthesis (DOS) has historically focused on the development of small-molecule collections with considerable chemical diversity with the hypothesis that chemical diversity will lead to diverse biological activities. We took a systematic approach to DOS to develop a focused library of reduced flavones from γ-pyrones with diversity of appendage, stereochemistry, and chemical properties to determine which features of small molecules are most predictive of biological performance diversity. The effects of these systematic modifications on biodiversity were determined using Cell Painting and cytotoxicity assays to compare the results of multiple methods of assessment. We observed that a greater fraction of sp3 hybridized atoms (fsp3) does not always lead to enhanced biodiversity, that stereochemistry and appendage diversity both contribute to biodiversity, and that lipophilicity of the pyrone class of compounds correlates with biodiversity. These results will contribute to the development of a general algorithm to predict which chemical features should be considered during the synthesis of DOS libraries to create biological performance-diverse collections of small molecules for probe and drug discovery.

Phyllanthusmin Derivatives Induce Apoptosis and Reduce Tumor Burden in High-Grade Serous Ovarian Cancer by Late-Stage Autophagy Inhibition.

High-grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with a need for new therapeutics. Many of the most widely used chemotherapeutic drugs are derived from natural products or their semi-synthetic derivatives. We have developed potent synthetic analogues of a class of compounds known as phyllanthusmins, inspired by natural products isolated from Phyllanthus poilanei Beille. The most potent analogue, PHY34, had the highest potency in HGSOC cell lines in vitro and displayed cytotoxic activity through activation of apoptosis. PHY34 exerts its cytotoxic effects by inhibiting autophagy at a late stage in the pathway, involving the disruption of lysosomal function. The autophagy activator, rapamycin, combined with PHY34 eliminated apoptosis, suggesting that autophagy inhibition may be required for apoptosis. PHY34 was readily bioavailable through intraperitoneal administration in vivo where it significantly inhibited the growth of cancer cell lines in hollow fibers, as well as reduced tumor burden in a xenograft model. We demonstrate that PHY34 acts as a late-stage autophagy inhibitor with nanomolar potency and significant antitumor efficacy as a single agent against HGSOC in vivo This class of compounds holds promise as a potential, novel chemotherapeutic and demonstrates the effectiveness of targeting the autophagic pathway as a viable strategy for combating ovarian cancer. Mol Cancer Ther; 17(10); 2123-35. ©2018 AACR.

Microwave-Assisted, Asymmetric Synthesis of 3-Amino-2,3-Dihydrobenzofuran Flavonoid Derivatives from Chalcones.

A route to access 3-amino-2,3-dihydrobenzofurans that utilizes microwave-assisted organic synthesis to rapidly generate analogues has been developed. The route begins with an acid-catalyzed, microwave-assisted aldol condensation to generate chalcone intermediates, followed by a Corey-Bakshi-Shibata reduction and subsequent Sharpless asymmetric epoxidation to access stereoisomeric epoxyalcohols. The final step is a one-pot, microwave-assisted, regioselective, acid-catalyzed epoxide opening with various amines followed by an intramolecular nucleophilic aromatic substitution reaction to generate the 3-amino-2,3-dihydrobenzofurans. This route provides ready access to stereochemically and structurally diverse analogues of these flavonoid scaffolds. Additionally, a pilot library was synthesized, and the biological activity diversity of the chalcones and dihydrobenzofurans was explored in human carcinoma cell lines.