Chemoproteomic Profiling Reveals that Anticancer Natural Product Dankastatin†B Covalently Targets Mitochondrial VDAC3.

Chlorinated gymnastatin and dankastatin alkaloids derived from the fungal strain Gymnascella dankaliensis have been reported to possess significant anticancer activity but their mode of action is unknown. These members possess electrophilic functional groups that can might undergo covalent bond formation with specific proteins to exert their biological activity. To better understand the mechanism of action of this class of natural products, we mapped the proteome-wide cysteine reactivity of the most potent of these alkaloids, dankastatin B, by using activity-based protein profiling chemoproteomic approaches. We identified a primary target of dankastatin B in breast cancer cells as cysteine C65 of the voltage-dependent anion-selective channel on the outer mitochondrial membrane VDAC3. We demonstrated direct and covalent interaction of dankastatin B with VDAC3. VDAC3 knockdown conferred hypersensitivity to dankastatin B-mediated antiproliferative effects in breast cancer cells, thus indicating that VDAC3 was at least partially involved in the anticancer effects of this natural product. Our study reveals a potential mode of action of dankastatin B through covalent targeting of VDAC3 and highlights the utility of chemoproteomic approaches in gaining mechanistic understanding of electrophilic natural products.

Chemoproteomic Profiling Reveals that Anti-Cancer Natural Product Dankastatin B Covalently Targets Mitochondrial VDAC3.

Chlorinated gymnastatin and dankastatin alkaloids derived from the fungal strain Gymnascella dankaliensis have been reported to possess significant anti-cancer activity but their mode of action is unknown. These members possess electrophilic functional groups that may undergo covalent bond formation with specific proteins to exert their biological activity. To better understand the mechanism of action of this class of natural products, we mapped the proteome-wide cysteine-reactivity of the most potent of these alkaloids, dankastatin B, using activitybased protein profiling chemoproteomic approaches. We identified a primary target of dankastatin B in breast cancer cells as cysteine C65 of the voltage-dependent anion selective channel on the outer mitochondrial membrane VDAC3. We demonstrated direct and covalent interaction of dankastatin B with VDAC3. VDAC3 knockdown conferred hyper-sensitivity to dankastatin B-mediated anti-proliferative effects in breast cancer cells indicating that VDAC3 was at least partially involved in the anti-cancer effects of this natural product. Our study reveals a potential mode of action of dankastatin B through covalent targeting of VDAC3 and highlight the utility of chemoproteomic approaches in gaining mechanistic understanding of electrophilic natural products.

Ligandability of E3 Ligases for Targeted Protein Degradation Applications.

Targeted protein degradation (TPD) using proteolysis targeting chimeras (PROTACs) and molecular glue degraders has arisen as a powerful therapeutic modality for eliminating disease-causing proteins from cells. PROTACs and molecular glue degraders employ heterobifunctional or monovalent small molecules, respectively, to chemically induce the proximity of target proteins with E3 ubiquitin ligases to ubiquitinate and degrade specific proteins via the proteasome. Whereas TPD is an attractive therapeutic strategy for expanding the druggable proteome, only a relatively small number of E3 ligases out of the >600 E3 ligases encoded by the human genome have been exploited by small molecules for TPD applications. Here we review the existing E3 ligases that have thus far been successfully exploited for TPD and discuss chemoproteomics-enabled covalent screening strategies for discovering new E3 ligase recruiters. We also provide a chemoproteomic map of reactive cysteines within hundreds of E3 ligases that may represent potential ligandable sites that can be pharmacologically interrogated to uncover additional E3 ligase recruiters.

Photo-Brook rearrangement of acyl silanes as a strategy for photoaffinity probe design.

Photoaffinity labeling (PAL) is a powerful tool for the identification of non-covalent small molecule-protein interactions that are critical to drug discovery and medicinal chemistry, but this approach is limited to only a small subset of robust photocrosslinkers. The identification of new photoreactive motifs capable of covalent target capture is therefore highly desirable. Herein, we report the design, synthesis, and evaluation of a new class of PAL warheads based on the UV-triggered 1,2-photo-Brook rearrangement of acyl silanes, which hitherto have not been explored for PAL workflows. Irradiation of a series of probes in cell lysate revealed an iPr-substituted acyl silane with superior photolabeling and minimal thermal background labeling compared to other substituted acyl silanes. Further, small molecule (+)-JQ1- and rapamycin-derived iPr acyl silanes were shown to selectively label recombinant BRD4-BD1 and FKBP12, respectively, with minimal background. Together, these data highlight the untapped potential of acyl silanes as a novel, tunable scaffold for photoaffinity labeling.

Deubiquitinase-targeting chimeras for targeted protein stabilization.

Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.

Chemical investigations into the biosynthesis of the gymnastatin and dankastatin alkaloids.

Electrophilic natural products have provided fertile ground for understanding how nature inhibits protein function using covalent bond formation. The fungal strain Gymnascella dankaliensis has provided an especially interesting collection of halogenated cytotoxic agents derived from tyrosine which feature an array of reactive functional groups. Herein we explore chemical and potentially biosynthetic relationships between architecturally complex gymnastatin and dankastatin members, finding conditions that favor formation of a given scaffold from a common intermediate. Additionally, we find that multiple natural products can also be formed from aranorosin, a non-halogenated natural product also produced by Gymnascella sp. fungi, using simple chloride salts thus offering an alternative hypothesis for the origins of these compounds in nature. Finally, growth inhibitory activity of multiple members against human triple negative breast cancer cells is reported.