Objective To establish an in vitro enzymatic activity evaluation system for OTULIN （OTU domain DUB with linear linkage specificity）and provide an experimental basis for OTULIN inhibitor screening. Methods The linear ubiquitin substrate protein His-Ub-Ub-Flag was designed, expressed, and purified. Using OTULIN^80-352 as the target enzyme and PR619 as a positive control, the enzyme-to-substrate ratio and reaction time were optimized to establish an in vitro OTULIN activity evaluation system. Results The linear ubiquitin substrate protein His-Ub-Ub-Flag was successfully obtained. OTULIN activity was significantly inhibited by PR619, which confirmed the feasibility of the assay. The optimal enzyme-to-substrate ratio of OTULIN^80-352 to His-Ub-Ub-Flag was 1∶16, and the optimal reaction condition was incubation at 37℃ for 20 min. Conclusion An in vitro enzymatic activity evaluation system for OTULIN was successfully established, which could be used to evaluate the inhibitory effects of candidate compounds on OTULIN activity and provide technical support for subsequent screening of OTULIN small-molecule inhibitors.

Membrane protein degradation is a cutting-edge field in targeted protein degradation (TPD). Herein, we developed glypican-3 (GPC3)-mediated lysosome-targeting chimeras (GLTACs) as a novel strategy for the targeted degradation of tumor-specific membrane proteins. GLTACs utilize tumor-specific expression and endocytosis properties of GPC3 to degrade membrane proteins. By conjugating a GPC3-targeting peptide with the ligand of protein of interest (POI), GLTACs induce the formation of a ternary complex that is internalized into lysosomes, leading to the degradation of the POI. The effectiveness and specificity of GLTACs were validated by designing PD-L1, c-Met, and FGFR1 degraders. In particular, GLTAC WP0 potently degraded PD-L1 and induced T-cell-mediated tumor killing against HepG2 cells, highlighting the potential therapeutic applications. The development of GLTAC technology expands the scope of TPD strategies and opens new avenues for discovering novel therapeutic modalities against challenging protein targets.

Objective To design and synthesize PROTAC degraders targeting the SARS-CoV-2 main protease （M^pro）based on PROTAC technology. Methods Compound 3w was used as the M^pro ligand, and the indole N atom in the solvent-exposed region was selected as the linker attachment site. A series of M^pro PROTACs were designed and synthesized by conjugating compound 3w with the CRBN ligand pomalidomide through alkane linkers of different lengths. The structures of the target compounds were confirmed by ¹H NMR, ¹³C NMR, and HRMS. Western Blot analysis was employed to evaluate their degradation activity and explore its mechanism in M^pro-HEK-293T cells. Results Four novel M^pro PROTACs（A1-A4）were successfully synthesized. The most potent compound A4 demonstrated M^pro degradation activity with a DC₅₀ value of 5.2 μmol/L, and its degradation mechanism was validated. Conclusion A novel class of M^pro PROTAC degraders were successfully designed and synthesized, and their protein degradation capability and mechanism of action were demonstrated. These results provided lead compounds for the research and development of antiviral degraders against SARS-CoV-2.

Objective To screen small molecule inhibitors of Fusobacterium nucleatum （Fn） based on commercially available compound libraries, and investigate their anti-colorectal cancer activities under Fn intervention in order to obtain novel anti-colorectal cancer lead compounds. Methods The promotion of colorectal cancer proliferation on organoid was validated by Fn. Secondly, the effects of anti-Fn compounds on their in vitro anticancer activity under Fn’s co-incubation with colorectal cancer HCT116 cell were comparative investigated. Finally, in vivo anticancer efficacy of highly active compounds on nude mouse colon cancer HCT116 transplanted tumor under the intervention of Fn was evaluated by gavage. Results Fn could significantly promote the proliferation of rectal cancer organoids. 9 anti-Fn active compounds could significantly enhance their in vitro anticancer activity under Fn’s co-incubation with HCT116 cells. Methotrexate had the strongest anti-cancer activity with IC₅₀ as 0.03 μmol/L. The combined use of methotrexate (0.5 mg/kg) and PD-1 (5.0 mg/kg) had a stronger anti-tumor effect than their standalone use. Conclusion As new small molecule inhibitor of Fn, methotrexate exhibited good in vitro and in vivo anti-colorectal cancer activity against HCT116 cells and nude mouse xenografts under Fn intervention, which showed the foundation for subsequent structural optimization, and could be expected to expand the new indications of methotrexate.

Ferroptosis,discovered in 2012,is a newly form of non-apoptotic and non-necrotic cell death,which is characterized by an increasement in lipid peroxidation and accumulation of intracellular iron ions.Glutathione peroxidase 4(GPX4)is the fourth member of the selenoprotein GPx family and plays a crucial role in clearing lipid peroxides in cells,making it an important regulator of ferroptosis.Small molecule inhibitors targeting GPX4 can induce ferroptosis,offering a new strategy for treating drug-resistant cancers and neurodegenerative diseases.The protein structure and function of GPX4 were primarily discusseed,and the latest advances in small molecule inhibitors of GPX4 were summarized,which provided a research foundation for developing ferroptosis inducers based on GPX4 inhibition.

Targeted protein degradation (TPD) techniques eliminate pathogenic proteins by hijacking the intracellular proteolysis machinery which includes the ubiquitin-proteasome system (UPS) and the lysosomal degradation pathway, holding promise to overcome the limitations of traditional inhibitors and further broaden the target space including many “undruggable” targets, and provide new targeted treatments for drug discovery. In this review, recent advances in a variety of promising TPD strategies were summarized, such as proteolysis targeting chimera (PROTAC), molecular glue, lysosome-targeting chimaera (LYTAC), autophagosome-tethering compound (ATTEC), autophagy-targeting chimera AUTAC and AUTOTAC, particularly. The representative case studies, potential applications and challenges were analyzed.

Objective To design and synthesize autophagic degraders targeting BRD4 based on autophagosome tethering compound (ATTEC) strategy and test their BRD4 degradation activity. Methods BRD4-targeting ATTECs were constructed by conjugating ispinesib that used as a LC3 ligand and JQ1 through a variety of alkane linkers. The final compounds were confirmed by ¹H NMR, ¹³C NMR and ESI-MS, and their degradation activity in different cell lines were tested by Western Blot. Results Five BRD4-ATTEC molecules were successfully synthesized for the first time. Compound 4 showed moderate BRD4 degradation activity in different cell lines. Conclusion The novel BRD4 autophagic degraders were discovered, which expanded the applicability of targeted autophagic degradation via ATTEC.