Uncovering PROTAC Sensitivity and Efficacy by Multidimensional Proteome Profiling: A Case for STAT3.

Proteolysis-targeting chimera (PROTAC) is a powerful technology that can effectively trigger the degradation of target proteins. The intricate interplay among various factors leads to a heterogeneous drug response, bringing about significant challenges in comprehending drug mechanisms. Our study applied data-independent acquisition-based mass spectrometry to multidimensional proteome profiling of PROTAC (DIA-MPP) to uncover the efficacy and sensitivity of the PROTAC compound. We profiled the signal transducer and activator of transcription 3 (STAT3) PROTAC degrader in six leukemia and lymphoma cell lines under multiple conditions, demonstrating the pharmacodynamic properties and downstream biological responses. Through comparison between sensitive and insensitive cell lines, we revealed that STAT1 can be regarded as a biomarker for STAT3 PROTAC degrader, which was validated in cells, patient-derived organoids, and mouse models. These results set an example for a comprehensive description of the multidimensional PROTAC pharmacodynamic response and PROTAC drug sensitivity biomarker exploration.

Thimerosal, a competitive thioredoxin reductase 1 (TrxR1) inhibitor discovered via high-throughput screening.

Thioredoxin reductase 1 (TrxR1) is considered as an important anti-cancer drug target, inhibition of which can induce reactive oxygen species (ROS)-mediated apoptosis of human cancer cells. Here, we developed and optimized a high-throughput screening (HTS) assay based on enzyme kinetics for the discovery of TrxR1 inhibitors. By utilizing this assay, we performed a HTS for 2500 compounds from an in-house library against TrxR1. We found that a vaccine preservative, thimerosal, strongly inhibited TrxR1 in a competitive and reversible manner with an IC50 of 24.08 ± 0.86 nM. In addition, we determined that thiomersal has an inhibitory effect on the proliferation of A549 lung cancer cell line, with a GI50 of 6.81 ± 0.09 µM, slightly more potent than auranofin (GI50 = 11.85 ± 0.56 µM). Furthermore, we showed by flow cytometer that thimerosal effectively increased the content of ROS in A549 cells. Therefore, our work provided a high-throughput screening assay to quickly and effectively discover TrxR1 inhibitors, identifying thiomersal as a novel TrxR1 inhibitor and chemical probe.

Rational Design of PDA/P-PVDF@PP Janus Membrane with Asymmetric Wettability for Switchable Emulsion Separation.

Water pollution caused by oil spills or sewage discharges has become a serious ecological environmental issue. Despite the membrane separation technique having a promising application in wastewater purification, the membrane fabrication method and separation robustness have remained unsatisfactory until now. Herein, we developed a novel strategy, spacer-assisted sequential phase conversion, to create a patterned polyvinylidene fluoride@polypropylene (P-PVDF@PP) substrate membrane with a multiscale roughened surface. Based on that surface structure, the underwater oil resistance behavior of the P-PVDF@PP membrane was improved. Moreover, owing to the abundant active sites on the P-PVDF@PP surface, the polydopamine/P-PVDF@PP (PDA/P-PVDF@PP) Janus membrane could be readily fabricated via wet chemical modification, which exhibited excellent switchable oil-water separation performance. Regarding surfactant-stabilized oil-water emulsion, the as-prepared PDA/P-PVDF@PP Janus membrane also had robust separation efficiency (as high as 99% in the n-hexane/water, chloroform/water, and toluene/water emulsion separation cases) and desirable reusability. Finally, the underlying mechanism of emulsion separation in the PDA/P-PVDF@PP Janus membrane was specified. The as-designed PDA/P-PVDF@PP Janus membrane with high-efficiency oil-water separation shows potential application in oily wastewater treatment, and the developed fabrication method has implications for the fabrication of advanced separation membranes.

Structure-Guided Development of Small-Molecule PRC2 Inhibitors Targeting EZH2-EED Interaction.

Disruption of EZH2-embryonic ectoderm development (EED) protein-protein interaction (PPI) is a new promising cancer therapeutic strategy. We have previously reported the discovery of astemizole, a small-molecule inhibitor targeting the EZH2-EED PPI. Herein, we report the cocrystal structure of EED in complex with astemizole at 2.15 Å. The structure elucidates the detailed binding mode of astemizole to EED and provides a structure-guided design for the discovery of a novel EZH2-EED interaction inhibitor, DC-PRC2in-01, with an affinity Kd of 4.56 µM. DC-PRC2in-01 destabilizes the PRC2 complex, thereby leading to the degradation of PRC2 core proteins and the decrease of global H3K27me3 levels in cancer cells. The proliferation of PRC2-driven lymphomas cells is effectively inhibited, and the cell cycle is arrested in the G0/G1 phase. Together, these data demonstrate that DC-PRC2in-01 could be an effective chemical probe for investigating the PRC2-related physiology and pathology and providing a promising chemical scaffold for further development.

Discovery and characterization of a novel glucose-6-phosphate dehydrogenase (G6PD) inhibitor via high-throughput screening.

Altered glucose-6-phosphate dehydrogenase (G6PD) status is influential in many cellular pathophysiological processes and diseases, making G6PD a potential target for cancer therapy. However, the available G6PD inhibitors are very limited and restricted. Here we developed a reducing equivalent nicotinamide adenine dinucleotide phosphate (NADPH) absorption photometry assay based on enzyme kinetics to characterize G6PD activity. In this way, we performed a high-throughput screening (HTS) to an in house library. And then we identified compound named Wedelolactone inhibiting G6PD strongly in a non-competitive, reversible way. In addition, we did the surface Plasmon Resonance (SPR) assay and indicated the KD between Wedelolactone and G6PD protein was 3.64 µM. Furthermore, our basic colony formation assay showed the inhibitory effect of Wedelolactone on the proliferation of ovarian cancer cells (IC50 ~ 10 µM). Thus, we provided a high-throughput screening assay to quickly and efficiently discover G6PD inhibitors, and identified Wedelolactone as a G6PD inhibitor, implying that Wedelolactone suppresses ovarian cancer partly through targeting G6PD.

Nano-Sized Cuboid-Shaped Phase in Mg-Nd-Y Alloy and its Behavior During Isothermal Aging.

In the present study, nano-sized cuboid-shaped particles in Mg-Nd-Y are studied by means of Cs-corrected atomic-scale high-angle annular dark-field scanning transmission electron microscopy. The structure of the cuboid-shaped phase is identified to be yttrium (major component) and neodymium atoms in face-centered cubic arrangement without the participation of Mg. The lattice parameter a=5.15 Å. During isothermal aging at 225°C, Mg3(Nd,Y) precipitates adhere to surface (100) planes of the cuboid-shaped particles with the orientation relationship: $[100]_{{{\rm Mg}_{{\rm 3}} {\rm RE}}} \,/\,\,/\,[100]_{{{\rm Cuboid}}} $ and $[310]_{{{\rm Mg}_{{\rm 3}} {\rm RE}}} \,/\,\,/\,[012]_{{{\rm Cuboid}}} $ . The fully coherent interfaces between the precipitates and the cuboid-shaped phases are reconstructed and categorized into two types: $(400)_{{{\rm Mg}_{{\rm 3}} {\rm RE}}} $ interface and $(200)_{{{\rm Mg}_{{\rm 3}} {\rm RE}}} $ interface.