Covalent Degrader of the Oncogenic Transcription Factor ß-Catenin.

ß-catenin (CTNNB1) is an oncogenic transcription factor that is important in cell-cell adhesion and transcription of cell proliferation and survival genes that drive the pathogenesis of many different types of cancers. However, direct pharmacological targeting of CTNNB1 has remained challenging. Here, we have performed a screen with a library of cysteine-reactive covalent ligands to identify the monovalent degrader EN83 that depletes CTNNB1 in a ubiquitin-proteasome-dependent manner. We show that EN83 directly and covalently targets CTNNB1 three cysteines C466, C520, and C619, leading to destabilization and degradation of CTNNB1. Through structural optimization, we generate a highly potent and relatively selective destabilizing degrader that acts through the targeting of only C619 on CTNNB1. Our results show that chemoproteomic approaches can be used to covalently target and degrade challenging transcription factors like CTNNB1 through destabilization-mediated degradation.

Ophiobolin A Covalently Targets Mitochondrial Complex IV Leading to Metabolic Collapse in Cancer Cells.

Ophiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anticancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of the OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets: lysine-72 of cytochrome c oxidase subunit 5A (COX5A) and cysteine-53 of mitochondrial hypoxia induced gene 1 domain family member 2A (HIGD2A). These two subunit proteins are part of complex IV (cytochrome C oxidase) within the electron transport chain and contributed significantly to the antiproliferative activity of OPA. OPA activated mitochondrial respiration in a COX5A- and HIGD2A-dependent manner, leading to an initial spike in mitochondrial ATP and heightened mitochondrial oxidative stress. OPA compromised mitochondrial membrane potential, ultimately leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anticancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.

Covalent Degrader of the Oncogenic Transcription Factor ß-Catenin.

ß-catenin (CTNNB1) is an oncogenic transcription factor that is important in cell-cell adhesion and transcription of cell proliferation and survival genes that drives the pathogenesis of many different types of cancers. However, direct pharmacological targeting of CTNNB1 has remained challenging deeming this transcription factor as "undruggable." Here, we have performed a screen with a library of cysteine-reactive covalent ligands to identify a monovalent degrader EN83 that depletes CTNNB1 in a ubiquitin-proteasome-dependent manner. We show that EN83 directly and covalently targets CTNNB1 through targeting four distinct cysteines within the armadillo repeat domain-C439, C466, C520, and C619-leading to a destabilization of CTNNB1. Using covalent chemoproteomic approaches, we show that EN83 directly engages CTNNB1 in cells with a moderate degree of selectivity. We further demonstrate that direct covalent targeting of three of these four cysteines--C466, C520, and C619--in cells contributes to CTNNB1 degradation in cells. We also demonstrate that EN83 can be further optimized to yield more potent CTNNB1 binders and degraders. Our results show that chemoproteomic approaches can be used to covalently target and degrade challenging transcription factors like CTNNB1 through a destabilization-mediated degradation.

Versatile flexible SERS substrate for in situ detection of contaminants in water and fruits based on Ag NPs decorated wrinkled PDMS film.

Flexible surface-enhanced Raman spectroscopy (SERS) substrate has attracted great attention due to its convenient sampling and on-site monitoring capability. However, it is still challenging to fabricate a versatile flexible SERS substrate, which can be used for in situ detection of analytes either in water or on irregular solid surfaces. Here, we report a flexible and transparent SERS substrate based on a wrinkled polydimethylsiloxane (PDMS) film obtained by transferring corrugated structures on the aluminium/polystyrene bilayer film, onto which silver nanoparticles (Ag NPs) are deposited by thermal evaporation. The as-fabricated SERS substrate exhibits a high enhancement factor (∼1.19×105), good signal uniformity (RSD of 6.27%), and excellent batch-to-batch reproducibility (RSD of 7.3%) for rhodamine 6 G. In addition, the Ag NPs@W-PDMS film can maintain high detection sensitivity even after mechanical deformations of bending or torsion for 100 cycles. More importantly, being flexible, transparent, and light, the Ag NPs@W-PDMS film can both float on the water surface and conformally contact with the curved surface for in situ detection. The malachite green in aqueous environment and on apple peel can be easily detected down to 10-6 M with a portable Raman spectrometer. Therefore, it is expected that such a versatile flexible SERS substrate has great potential in on-site, in situ contaminant monitoring for realistic applications.

Ag NPs@PDMS nanoripple array films as SERS substrates for rapid in situ detection of pesticide residues.

The large-area fabrication of flexible and transparent surface-enhanced Raman scattering (SERS) substrates with high performance by a facile and efficient method is still challenging. Here, we demonstrated a large-scale, flexible and transparent SERS substrate composed of PDMS nanoripple array film decorated with silver nanoparticles (Ag NPs@PDMS-NR array film) prepared by a combination of plasma treatment and magnetron sputtering. The performances of SERS substrates were characterized by rhodamine 6G (R6G) using a handheld Raman spectrometer. The optimal Ag NPs@PDMS-NR array film exhibited high SERS sensitivity, with a detection limitation of R6G reaching 8.20 × 10-8 M as well as excellent uniformity (RSD = 6.8%) and batch-to-batch reproducibility (RSD = 2.3%). In addition, the substrate showed outstanding mechanical stability and good SERS enhancement by backside illumination, thus it was suitable for in situ SERS detection on curved surfaces. The detection limit of malachite green on apple and tomato peels was 1.19 × 10-7 and 1.16 × 10-7 M, respectively, and quantitative analysis of pesticide residues could be realized. These results demonstrate that the Ag NPs@PDMS-NR array film has great practical potential in rapid in situ detection of pollutants.

Ophiobolin A Covalently Targets Complex IV Leading to Mitochondrial Metabolic Collapse in Cancer Cells.

Ophiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anti-cancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets-cysteine C53 of HIG2DA and lysine K72 of COX5A-that are part of complex IV of the electron transport chain and contributed significantly to the anti-proliferative activity. OPA activated mitochondrial respiration in a HIG2DA and COX5A-dependent manner, led to an initial spike in mitochondrial ATP, but then compromised mitochondrial membrane potential leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anti-cancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.

Homoploid F1 hybrids and segmental allotetraploids of japonica and indica rice subspecies show similar and enhanced tolerance to nitrogen deficiency than parental lines.

It remains unclear whether the merger of two divergent genomes by hybridization at the homoploid level or coupled with whole-genome duplication (WGD; allopolyploidy) can result in plants having better tolerance to stress conditions. In this study, we compared phenotypic performance and gene expression in the two diploid subspecies of rice (Oryza sativa subsp. japonica and indica), their reciprocal F1 hybrids, and in segmental allotetraploids under normal and nitrogen (N)-deficient conditions. We found that F1 hybrids and tetraploids showed higher and similar levels of tolerance to N deficiency than either parent. In parallel, total expression levels of 18 relevant functional genes were less perturbed by N deficiency in the F1 hybrids and tetraploids than in the parents. This was consistent with stable intrinsic partitioning of allelic/homoeologous expression defined by parental legacy in the homoploid F1 hybrids/tetraploids between the two conditions. The results suggest that genetic additivity at both the homoploid and allopolyploidy level might lead to similar beneficial phenotypic responses to nitrogen stress compared with the parents. The lack of synergistic responses to N limitation concomitant with WGD, relative to that exhibited by F1 hybrids, adds new empirical evidence in support of the emerging hypothesis that hybridization by itself can play a significant role in plant adaptive evolution in times of stress.

Identifying potential serum biomarkers of breast cancer through targeted free fatty acid profiles screening based on a GC-MS platform.

Recent advances suggest that abnormal fatty acid metabolism highly correlates with breast cancer, which provide clues to discover potential biomarkers of breast cancer. This study aims to identify serum free fatty acid (FFA) metabolic profiles and screen potential biomarkers for breast cancer diagnosis. Gas chromatography-mass spectrometry and our in-house fatty acid methyl ester standard substances library were combined to accurately identify FFA profiles in serum samples of breast cancer patients and breast adenosis patients (as controls). Potential biomarkers were screened by applying statistical analysis. A total of 18 FFAs were accurately identified in serum sample. Two groups of patients were correctly discriminated by the orthogonal partial least squares-discriminant analysis model based on FFA profiles. Seven FFA levels were significantly higher in serum from breast cancer patients than that in controls, and exhibited positive correlation with malignant degrees of disease. Furthermore, five candidates (palmitic acid, oleic acid, cis-8,11,14-eicosatrienoic acid, docosanoic acid and the ratio of oleic acid to stearic acid) were selected as potential serum biomarkers for differential diagnosis of breast cancer. Our study will help to reveal the metabolic signature of FFAs in breast cancer patients, and provides valuable information for facilitating clinical noninvasive diagnosis.