Discovery and Biological Evaluation of a Novel Small Molecule Cbl-b Inhibitor

Discovery and Biological Evaluation of a Novel Small Molecule Cbl-b Inhibitor | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 24 January 2025 V1 Latest version Share on Discovery and Biological Evaluation of a Novel Small Molecule Cbl-b Inhibitor Authors : Wei He , Zisheng Fan , Manlin Huang , Min Wu , Zhiming Ge , Jie Yu , Yuanyang Zhou , Jiahang Xu , Mingyue Zheng , and Sulin Zhang 0000-0002-9167-4689 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173769856.61608204/v1 355 views 238 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Abstract Background and Purpose: Cbl-b, a RING-type E3 ubiquitin ligase in the Casitas B-lineage lymphoma protein family, negatively regulates immune responses. Its inhibition enhances immune cell function, aiding in combating pathogens and tumors. This study aimed to discover novel small-molecule Cbl-b inhibitors and evaluate their potential in modulating immune activity. Experimental approach: A high-throughput screening of a self-constructed compound library identified hit compounds. Their interaction with Cbl-b was validated at the molecular level, and molecular docking confirmed binding sites. In vitro phosphorylation and ubiquitination systems were used to explore the inhibitory mechanism of 450F10, with cellular activity assessed in Jurkat cells and CD8+ T cells. Key Results: It was demonstrated that 450F10 binds specifically to F263 and Y260 of Cbl-b, thus inhibiting the phosphorylation-dependent activation of Cbl-b, thereby suppressing its protein activity. Notably, It enhances interleukin-2 (IL-2) secretion by human peripheral CD8+ T cells, boosting immune responses. Conclusion and implications: This study identified 450F10 as a novel small-molecule Cbl-b inhibitor capable of enhancing TCR signal stimulation and promoting IL-2 secretion in CD8+ T cells without affecting cell proliferation at effective concentrations. 450F10 shows potential as a valuable tool for exploring Cbl-b’s role in immune regulation. Future efforts will focus on optimizing the compound’s structure to enhance its activity and evaluating its efficacy and safety in animal models. K E Y W O R D S: Cbl-b, High-Throughput Screening, T Cell Activation, Small Molecule Inhibitors, Immunotherapy Discovery and Biological Evaluation of a Novel Small Molecule Cbl-b Inhibitor Wei He 1,2 # , Zisheng Fan 3,5 # , Manlin Huang 1,2 , Min Wu 1,2 , Zhiming Ge 2,6,7 , Jie Yu 4,5 , Yuanyang Zhou 3,5 , Jiahang Xu 2,6,7 , Mingyue Zheng 1,2,3,4,5,6,7 * , Sulin Zhang 2,7 * 1. School of Pharmacy, Nanchang University, Nanchang 330031, China. 2. Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. 3. Shanghai Institute for Advanced Immunochemical Studies, and School of Life Science and Technology, ShanghaiTech University, Shanghai, China. 4. School of Information Science and Technology, Shanghai Tech University, Shanghai, 201210, China. 5. Lingang Laboratory, Shanghai 200031, China 6. School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China. 7. University of Chinese Academy of Sciences, Beijing, 100049, China. # Wei He and Zisheng Fan contributed equally. * Corresponding authors E-mail addresses: [email protected] (S. Zhang), [email protected] (M. Zheng) Abstract Background and Purpose: Cbl-b, a RING-type E3 ubiquitin ligase in the Casitas B-lineage lymphoma protein family, negatively regulates immune responses. Its inhibition enhances immune cell function, aiding in combating pathogens and tumors. This study aimed to discover novel small-molecule Cbl-b inhibitors and evaluate their potential in modulating immune activity. Experimental approach: A high-throughput screening of a self-constructed compound library identified hit compounds. Their interaction with Cbl-b was validated at the molecular level, and molecular docking confirmed binding sites. In vitro phosphorylation and ubiquitination systems were used to explore the inhibitory mechanism of 450F10, with cellular activity assessed in Jurkat cells and CD8 + T cells. Key Results: It was demonstrated that 450F10 binds specifically to F263 and Y260 of Cbl-b, thus inhibiting the phosphorylation-dependent activation of Cbl-b, thereby suppressing its protein activity. Notably, It enhances interleukin-2 (IL-2) secretion by human peripheral CD8 + T cells, boosting immune responses. Conclusion and implications: This study identified 450F10 as a novel small-molecule Cbl-b inhibitor capable of enhancing TCR signal stimulation and promoting IL-2 secretion in CD8 + T cells without affecting cell proliferation at effective concentrations. 450F10 shows potential as a valuable tool for exploring Cbl-b’s role in immune regulation. Future efforts will focus on optimizing the compound’s structure to enhance its activity and evaluating its efficacy and safety in animal models. K E Y W O R D S: Cbl-b, High-Throughput Screening, T Cell Activation, Small Molecule Inhibitors, Immunotherapy What is already known？ Cbl-b is a negative regulator of immune cells, and inhibiting Cbl-b can promote T cell activation and enhance immune function. What does this study add？ A novel Cbl-b inhibitor, 450F10, was identified through high-throughput screening, and its inhibitory activity and mechanism were studied. 450F10 enhances IL-2 secretion in CD8 + T cells, improving immune responses. What is the clinical significance？ This study provides new impetus and support for discovering novel Cbl-b inhibitors and advancing them toward clinical application. 1. Introduction Since the discovery of immune checkpoints (ICIs), immunotherapies that enhance immune cell activity by inhibiting immune checkpoints, such as PD-1/PD-L1 and CTLA-4 monoclonal antibodies, have garnered widespread attention (Q. Li, Han, Yang, & Chen, 2022) (Ghahremanloo, Soltani, Modaresi, & Hashemy, 2019) and achieved remarkable clinical outcomes (Nishijima, Muss, Shachar, & Moschos, 2016). However, with the extensive application of ICIs, their limitations and challenges have become apparent. These include reliance on reliable biomarker screening (X. Li, Shao, Shi, & Han, 2018), severe adverse effects (Y. Liu & Zheng, 2020) (Poto et al., 2022) (Dong et al., 2022), resistance with prolonged use (Shi, Lan, & Yang, 2020), and variability in efficacy among individuals (X. Li et al., 2018) (Shi et al., 2020). These issues pose significant challenges for the clinical application of ICIs. To address these limitations, increasing efforts are being devoted to identifying novel molecular targets that can further enhance the immune system’s ability to eliminate tumors effectively. Protein ubiquitination mediated by E3 ubiquitin ligases plays a crucial role in regulating innate and adaptive immunity (Bhoj & Chen, 2009). Cbl-b, a member of the Casitas B-lineage lymphoma protein family, functions as an E3 ubiquitin ligase and is vital in various immune cells, including NK cells, B cells, and T cells (Tang, Langdon, & Zhang, 2019). As a negative feedback regulator of immune function, Cbl-b prevents excessive immune responses that could lead to autoimmune diseases (Jeon et al., 2004) (Venuprasad, 2010). Studies have shown that Cbl-b is critical in regulating T-cell activity (Tang et al., 2019) (Schmitz, 2009). Upon T-cell activation, the T-cell receptor (TCR) signaling pathway induces phosphorylation of downstream proteins such as PLCγ1 and Vav1. p-PLCγ1 activates the TCR signaling cascade, promoting T-cell activation and cytokine secretion, and p-Vav1 optimizes TCR complex assembly, enhancing signal transduction. These processes collectively facilitate the release of immune mediators and transcription factors, increasing IL-2 secretion and stimulating immune activity (Augustin, Bao, & Luke, 2023). Simultaneously, phosphorylation of Tyrosine 363 (Y363) on Cbl-b activates its E3 ligase function, ubiquitinating and degrading phosphorylated PLCγ1 and Vav1, thereby inhibiting Immune factors release and T-cell activation (Schmitz, 2009) (Augustin et al., 2023). Knockout studies of Cbl-b have revealed its significant role in immune modulation. For instance, knocking out Cbl-b prevents CD8 + T-cell exhaustion and enhances CAR-T cell function (Kumar et al., 2021). Cbl-b deletion in dendritic cells results in increased hepatic immune infiltration (Xu et al., 2022). Additionally, Cbl-b knockout CD8 + T cells exhibit innate resistance to spontaneous tumors(Loeser et al., 2007). Similarly, Cbl-b knockout mice show innate resistance to both transplanted and spontaneous tumors (Chiang, Jang, Hodes, & Gu, 2007). These findings suggest that Cbl-b is a promising target for immune modulation and tumor immunotherapy. In recent years, with the continuous advancement of drug screening platforms and technologies, an increasing number of studies have focused on the design and discovery of Cbl-b inhibitors, with several promising candidate compounds emerging from basic research (Augustin et al., 2023) (Zhou et al., 2024). For example, APN-401, developed by Alopexx Oncology, is one of the first gene-silencing inhibitors targeting Cbl-b. It uses siRNA technology to suppress Cbl-b expression, establishing Cbl-b as an immune modulation target, and is currently in Phase I clinical trials. In addition, NX-1607, developed by Nurix, is the first small-molecule Cbl-b inhibitor to enter clinical stages. It has progressed to Phase II and shows significant clinical translational potential. DeTIL-0255, also developed by Nurix, is an autologous cell therapy currently in Phase I clinical trials that expands tumor-infiltrating lymphocytes (TILs) extracted from a patient’s tumor in vitro , in combination with recombinant interleukin-2 (IL-2) and the small molecule Cbl-b inhibitor NX-0255. Furthermore, Cbl-b inhibitors developed by AstraZeneca and Simcere Pharmaceutical Group are also undergoing clinical research. Despite the great potential of targeting Cbl-b for tumor immunotherapy, there are currently few reported Cbl-b inhibitors, and the latest developments are still in the early clinical stages, with no inhibitors yet approved for market use. Besides, there is a lack of systematic clinical data to verify their long-term efficacy and safety. Therefore, the continuous discovery and optimization of new small-molecule Cbl-b inhibitors is crucial. Through innovative screening and optimization strategies, if inhibitors with better efficacy, stronger selectivity, or new mechanisms of action can be developed, it will not only help overcome current research bottlenecks but also provide more options for clinical development, injecting new momentum into the field of tumor immunotherapy. Cbl-b protein consists of three primary domains: a highly conserved N-terminal Tyrosine kinase binding (TKB) domain, a Linker helix region (LHR), and a RING domain (Thien & Langdon, 2001) ( Figure 1 a). The TKB domain comprises a four-helix bundle (4H), a calcium-binding EF-hand motif, and a modified Src homology 2 (SH2) domain, which collectively facilitate Tyrosine phosphorylation (Ohno et al., 2016). There is a tyrosine at position 363 between the TKB domain and LHR, and its phosphorylation is critical for the E3 ubiquitin ligase activity of Cbl-b. Structural studies using NMR and small-angle X-ray scattering have shown that, under normal conditions, the unphosphorylated N-terminal tail of Cbl-b adopts a compact conformation through intramolecular interactions, masking the interaction surface between the RING domain and E2 ubiquitin-conjugating enzyme, rendering Cbl-b inactive (Levkowitz et al., 1999) (Kobashigawa et al., 2011). Upon phosphorylation of Y363 in the linker region, the E2-binding surface of the RING domain is exposed, activating Cbl-b’s E3 ligase function to ubiquitinate downstream target proteins (Zheng, Wang, Jeffrey, & Pavletich, 2000) (Dou et al., 2012) (Figure 1b), thereby suppressing immune cell activity. Based on this background, inhibiting Cbl-b phosphorylation to maintain its inactive state could indirectly enhance immune cell activity and activate the immune system. Figure 1. Schematic representation of the domain structure of Cbl-b protein (a) and the principle of activation by Y363 phosphorylation of the Cbl-b protein (b). 2. Methods 2.1 Recombinant protein expression and purification To express and purify Cbl-b protein, the gene encoding its C-terminal domain (36-427) was cloned into the pET28a vector. Plasmids His-Cbl-b, GST-Cbl-b, and GST-Cbl-b-Flag were constructed using a 6×-His tag, GST tag, or a combination of GST and Flag tags, along with appropriate restriction sites. Similarly, UBA1 (41-1058), UbCH5b (full-length), and Zap70 (1-606) were cloned into the pET28a vector, while Ub (full-length) was cloned into the pGEX 6p-1 vector to create His-UBA1, His-UbCH5b, His-Zap70, and GST-Ub plasmids. Since expressing the Src gene in E. coli causes cell death, co-transfection with the yoPH gene was used to mitigate Src toxicity. A plasmid encoding Src (251-533) with a 6×-His tag was constructed in pET28a (His-Src), while yoPH was cloned into pCDFDuet-1 to create the yoPH plasmid. All proteins were expressed in E. coli BL21 (DE3) (Shanghai Weidi Biotechnology Co., Ltd.). For His-Cbl-b, recombinant E. coli was cultured in 1 L of LB medium at 37℃ for 4 hours. When OD of LB medium exceeded 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG, BBI#A600168-0100) was added, and expression was induced at 16℃ for 16 hours. Optimal IPTG concentrations were 1 mM for His-Cbl-b, GST-Cbl-b, GST-Cbl-b-Flag, and His-UbCH5b; 0.5 mM for GST-Ub and His-Zap70; and 0.2 mM and 0.1 mM for His-Src and His-UBA1, respectively. After induction, cells were harvested by centrifugation at 3000 rpm for 30 minutes and stored at -80℃. Proteins were purified using optimized buffer systems and strategies to ensure high purity and stability. The cell lysis buffer comprised buffer A supplemented with 1 mM TCEP and 1 mM PMSF. His-Cbl-b, His-UbCH5b, His-Src, and His-Zap70 shared the same buffer system: Buffer A (50 mM Hepes, pH 8.0, 300 mM NaCl, 20 mM Imidazole) and Buffer B (50 mM Hepes, pH 8.0, 300 mM NaCl, 300 mM Imidazole). Lysates were purified using HisTrap FF columns (GE Healthcare), eluted with buffer B, and further refined with Superdex75 10/300GL columns (GE Healthcare). Final storage used a system buffer (20 mM Hepes, pH 8.0, 200 mM NaCl). His-Src and His-UbCH5b underwent TEV protease treatment to remove the His tag, followed by further purification into low-salt buffer for experiments. For UBA1 protein, given its large molecular weight, poor solubility, and weak binding, buffer components were adjusted: Buffer A (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10% glycerol, 10 mM Imidazole) and Buffer B (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10% glycerol, 300 mM Imidazole). Purification and cleavage followed similar protocols. For GST-Ub and GST-Cbl-b-Flag, the same buffer system was used: Buffer A (50 mM Hepes, pH 8.0, 300 mM NaCl) and Buffer B (50 mM Hepes, pH 8.0, 300 mM NaCl, 10 mM Glutathione). Proteins were eluted from GSTrap FF columns (GE Healthcare) using buffer B, desalted, and exchanged into the GST cleavage buffer (Buffer A). After adding pp protease, the mixture was incubated at 4℃ overnight to remove the GST tag. The next day, the cleaved mixture was passed through a GSTrap FF column, further purified with a Superdex75 10/300GL column, and exchanged into low-salt system buffer (20 mM Hepes, pH 8.0, 200 mM NaCl) for storage. For HTRF experiments requiring the GST tag on GST-Ub protein, the cleavage step was omitted, and the protein was directly purified and stored. 2.2 Fluorescence Polarization Assay The Fluorescence Polarization (FP) assay were used to screen compounds that competitively inhibit the binding of Cbl-b protein to the modified probe. The experiment was conducted in opaque 384-well black plates (Corning#3575). Each well received 2 μL of compound and 19 μL of His-Cbl-b protein (final concentration of 250 nM). The mixture was incubated at room temperature (RT) for 30 minutes, after which 19 μL of probe (final concentration of 20 nM) was added to each well, and the incubation continued at RT for an additional 30 minutes. The fluorescence polarization signal was measured using a PE instrument (Revvity). Cbl-b-IN-1 was used as a positive control. 2.3 Protein thermal shift assay The Protein thermal shift assay (PTS) experiment was performed in a 96-well non-skirted PCR plate (DN Biotech#5371012) with a total reaction volume of 20 μL. Each reaction consisted of 19 μL of a mixture (3 μM purified protein, 5× SYPRO Orange (Invitrogen#S6651), and PTS buffer (20 mM HEPES, pH 8.0, 200 mM NaCl)) and 1 μL of compound. The PCR plate was placed in a CFX96 TM Real-Time PCR Detection System (Bio-Rad), and fluorescence was monitored between 25℃ and 90℃. The melting temperature ( T m ) of His-Cbl-b protein was determined using Bio-Rad software. Finally, the data were replotted using GraphPad 8.0. 2.4 Nuclear magnetic resonance Carr-Purcell-Meiboom-Gill (CPMG) and Saturation Transfer Difference (STD) NMR experiments were used to study the interaction between the compound and the protein. All NMR spectra were acquired at 25℃ using a Bruker Avance III-600 MHz spectrometer. The compounds (200 μM), dissolved in deuterated DMSO, and the protein His-Cbl-b (5 μM), diluted in deuterated PBS, were mixed at a 40:1 ratio and dissolved in phosphate-buffered saline (20 mM Tris-HCl, pH 8.0, 200 mM NaCl) for NMR data acquisition. 2.5 Surface plasmon resonance The surface plasmon resonance (SPR) binding assays were conducted at 25℃ using a Biacore 1K instrument (Cytiva). Purified His-Cbl-b protein and His-Cbl-b (Y260F/F263L) mutant protein were captured on a CM5 sensor chip (carboxymethylated dextran surface) using acetate buffer (pH 4.0). All experiments were performed in HBS running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl). The compound 450F10 was flowed over the sensor chip at a rate of 30 μL/min, with a binding time of 120 seconds and a dissociation time of 300 seconds. All binding analyses were performed using low molecular dynamics protocols. The data were fitted to a 1:1 binding model using Biacore 1K Evaluation Software (Cytiva), and the equilibrium dissociation constant ( K D ) was determined. 2.6 Isothermal titration calorimetry binding assay Isothermal titration calorimetry (ITC) was performed using a MicroCal PEAQ-ITC calorimeter to determine the binding parameters between 450F10 and His-Cbl-b wild-type protein as well as His-Cbl-b (Y260F/F263L) mutant protein. All experiments were conducted at 25℃ in titration buffer containing 20 mM HEPES (pH 8.0) and 200 mM NaCl. Recombinant protein (10 µM) was placed in the sample cell, and 450F10 (100 µM) was titrated into the sample in 20 consecutive 2 µL injections (the initial injection was 0.4 µL). Each injection was allowed to incubate for 120 seconds, and the dissociation constant ( K D ) for the interaction between 450F10 and His-Cbl-b protein and His-Cbl-b (Y260F/F263L) mutant protein was determined. Data were collected and analyzed using MicroCal PEAQ-ITC software. 2.7 In vitro phosphorylation assay The in vitro phosphorylation assay was designed to simulate the activation of Cbl-b protein by Src and to investigate whether compound 450F10 inhibits the phosphorylation of Cbl-b. In a PCR tube, 1 μL of compound and 9 μL of His-Cbl-b protein (diluted to a final concentration of 200 nM) in in vitro phosphorylation buffer (125 mM Hepes, pH 8.0, 10 mM MgCl 2 , 10 mM ATP, 2 mM DTT) were added. After incubating at RT for 30 minutes, Src protein (diluted to a final concentration of 100 nM) in the same phosphorylation buffer was added to induce Tyrosine phosphorylation at residue 363 of His-Cbl-b protein, followed by a 30-minute incubation at RT. The results were detected by Western Blot, using anti-pTYR antibody to detect phosphorylated Cbl-b levels, and anti-His-tag antibody as an internal control to measure total Cbl-b protein. 2.8 In vitro Ubiquitination assay The in vitro ubiquitination assay was designed to study the effect of compound 450F10 on the ubiquitination activity of Cbl-b protein. After incubation with the compound, Cbl-b protein is phosphorylated and activated by Src, and then incubated with E1 (UBA1), E2 (UbCH5b), and Ub proteins to simulate the in vivo ubiquitination process of the E3 ligase. Specifically, 1 μL of compound and 9 μL of GST-Cbl-b protein (diluted to a final concentration of 250 ng/μL) in ubiquitination buffer (20 mM Hepes, pH 8.0, 50 mM KCl, 10 mM MgCl 2 , 10 mM ATP, 2 mM DTT, 0.5 mg/mL Creatine Kinase (CK)) were mixed and incubated at RT for 30 minutes. Src protein (diluted to a final concentration of 50 ng/μL) was then added to activate GST-Cbl-b, followed by incubation for another 30 minutes. Finally, a mixture containing E1 (UBA1), E2 (UbCH5b), and Ub (final concentrations of 12.5 ng/μL, 25 ng/μL, and 25 ng/μL, respectively) was added, and the incubation was continued for 30 minutes. After sample preparation, ubiquitination levels of GST-Cbl-b were detected by Western Blot, using anti-Ub antibody to detect ubiquitination of Cbl-b and anti-GST antibody as an internal control to measure total Cbl-b protein. 2.9 Homogeneous time-resolved fluorescence The homogeneous time-resolved fluorescence (HTRF) assay was performed in a 384-well opaque white plate (Revvity#6007290). The magnetic beads used for HTRF, His-EU-Gold (61HI2KLA), Flag-XL665 (61FG2XLA), His-Tb (61HISTLF), and GST-XL665 (61GSTXLF), were all purchased from Revvity. The reaction system for the E2 (His-UbCH5b) - E3 (Cbl-b-Flag) binding assay was as follows: 1 μL of compound was added to 9 μL of Flag-tagged Cbl-b (diluted to a final concentration of 200 nM) in HTRF buffer (50 mM Hepes, pH 8.0, 100 mM NaCl, 10 mM ATP, 10 mM MgCl 2 , 0.1% BSA, 0.01% Triton X-100), and incubated at RT for 30 minutes. Then, 5 μL of Src (diluted to a final concentration of 600 nM) in the same HTRF buffer was added, followed by incubation at RT for 2 hours. Finally, 5 μL of a mix prepared in HTRF buffer (6×-His-UbCH5b (200 nM), His-EU-Gold (1:100), Flag-XL665 (1:200)) was added, and the mixture was incubated at RT for 30 minutes. The microplate was analyzed using a PE instrument (Revvity), with the excitation wavelength set to 340 nm and the emission intensities measured at 620 nm and 665 nm. The proximity of His-EU Gold-labeled proteins and Flag-XL665-labeled proteins was assessed by calculating the 665 nm/620 nm ratio, which is proportional to the degree of their binding. The strength of the fluorescence signal reflects the binding strength between His-UbCH5b and Cbl-b-Flag. The ATP-free group was used as a reference. The system for the HTRF assay of Cbl-b ubiquitination of Zap70 is similar to the E2-E3 binding assay, but with different proteins and magnetic beads. The reaction system is as follows: 1 μL of compound is added to 9 μL of Cbl-b (diluted to a final concentration of 200 nM) in HTRF buffer (50 mM Hepes, pH 8.0, 100 mM NaCl, 10 mM ATP, 10 mM MgCl 2 , 0.1% BSA, 0.01% Triton X-100), and incubated at RT for 30 min. Then, 5 μL of Src (diluted to a final concentration of 600 nM) is added, followed by incubation at RT for 2 hours. Finally, 5 μL of a mix prepared in HTRF buffer (His-Zap70 (200 nM), UbCH5b (200 nM), GST-Ub (200 nM), UBA1 (200 nM), His-Tb (1:100), GST-XL665 (1:100)) is added, and the mixture is incubated at RT for 30 minutes. The microplate was analyzed using a PE instrument (Revvity), with the excitation wavelength set to 340 nm and the emission intensities measured at 620 nm and 665 nm. The proximity of His-Tb-labeled proteins and GST-XL665-labeled proteins was assessed by calculating the 665 nm/620 nm ratio, which is proportional to the degree of their binding. The strength of the fluorescence signal reflects the amount of GST-Ub ubiquitination transferred to His-Zap70. The ATP-free group serves as the reference. 2.10 Cell lines Jurkat cells (WuhanPricella Biotechnology#CL0315) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, at 37℃ in a 5% CO 2 atmosphere. Human CD8 + T cells (Shanghai HeYoushengBio) were thawed the day before the experiment and cultured in RPMI 1640 medium containing 10% heat-inactivated FBS and 1% penicillin-streptomycin, at 37℃ in a 5% CO 2 atmosphere. 2.11 Cellular Thermal Shift Assay The Cellular Thermal Shift Assay (CETSA) was used to verify the binding of compound 450F10 to full-length Cbl-b protein in Jurkat cells. Jurkat cells were harvested and diluted to a concentration of 1 × 10 6 cells/mL with fresh complete medium. The cells were then evenly distributed into two groups, each with 10 mL. 450F10 (10 μM) was added to one group, and an equal volume of DMSO was added to the other group. Both groups were incubated for 1 hour at 37℃ in a 5% CO 2 incubator. After incubation, the cells were centrifuged to remove the supernatant, resuspended in PBS containing a protease inhibitor (Meilunbio#MB2678), and divided into 10 equal aliquots. These aliquots were heated for 3 minutes at temperatures ranging from 45℃ to 65℃ using a gradient heating program in a PCR machine, as well as at RT (25℃). Finally, the samples were lysed by repeated freeze-thaw cycles in liquid nitrogen, and protein stability at different temperatures in both the treatment and control groups was assessed by Western blot. 2.12 pY783-PLCγ1 Accumulation Assay The pY783-PLCγ1 accumulation assay was used to evaluate the effect of compound 450F10 on the downstream signaling of Cbl-b at the cellular level. Jurkat cells were seeded in a 12-well plate at a density of 5 × 10 5 cells per well and treated with different concentrations of compound. The cells were incubated at 37℃ in a 5% CO 2 incubator for 15 minutes. Subsequently, 6 μL of human T cell activator CD3/CD28 Dynabeads (Thermo Fisher Scientific#11131D) were added to each well to stimulate the cells, which were then incubated for 1 hour. Finally, the level of PLCγ1 phosphorylation at the pY783 site was assessed by Western blot analysis at the different compound concentrations. 2.13 Western Blotting All protein samples were lysed using RIPA lysis buffer (Beyotime#P0013C) containing phosphatase inhibitors (Bimake#B15001) and protease inhibitors (Bimake#B14001). Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific#23227). Equal amounts of protein (10 μg) were separated by 10% SDS-PAGE and transferred to a nitrocellulose (NC) membrane. The membrane was blocked for 1 hour in TBST buffer containing 5% skim milk (BD#232100), followed by overnight incubation with primary antibodies at 4℃. The next day, the membrane was incubated with the corresponding secondary antibodies at RT for 2 hours, then treated with ECL reagent (Meilunbio#MA0186) and visualized using a GeneGnome XRQ NPC system (Shanghai, China). Data were processed and quantitatively analyzed using ImageJ and GraphPad 8.0 software. The primary antibodies used for protein immunoblotting were as follows: anti-Cbl-b (CST#9498S, 1:1000), anti-p-PLCγ1 (Y783) (CST#14008, 1:1000), anti-PLCγ1 (Abclonal#A8899, 1:1000), anti-β-Tubulin (Abclonal#AC015, 1:1000), anti-pY (SantaCruz#sc-508, 1:1000), anti-His-tag (Proteintech#66005-1-Ig, 1:1000), anti-GST-tag (Abclonal#66001-2-lg, 1:1000), anti-Ub (CST#3933S, 1:1000), and anti-β-actin (Abclonal#AC038, 1:1000). 2.14 Flow Cytometry Flow cytometry was used to analyze the effect of compound 450F10 on the secretion of IL-2 in human peripheral CD8 + T cells. Human peripheral CD8+ T cells were seeded at a density of 1 × 10 5 cells per well in a 96-well plate and cultured overnight at 37℃ in a 5% CO 2 incubator. The cells were then treated with 5 μM of compound 450F10, Cbl-b-IN-1 (or DMSO) for 30 minutes at 37℃ in a 5% CO 2 incubator. After treatment, 2 μL of human T cell activator CD3/CD28 Dynabeads (Thermo Fisher Scientific#11131D) were added to each well to stimulate the CD8 + T cells, which were incubated for 24 hours at 37℃ in a 5% CO 2 incubator. The cells were then centrifuged, and the supernatant was removed. Cells were washed three times with PBS and then fixed with 100 μL of fixation buffer (Thermo Fisher Scientific#00-8222-49) at 4℃ for 40 minutes. Subsequently, 200 μL of permeabilization buffer (Thermo Fisher Scientific#00-8333-56) was added to each well, followed by centrifugation to remove the supernatant, followed by washing with permeabilization buffer three times. The cells were then incubated with 5 μL each of BV421-CD3 antibody (Thermo Fisher Scientific#404-0037-42) and PE-IL2 antibody (Thermo Fisher Scientific#12-7029-42) for 40 minutes at 4℃. After incubation, 200 μL of permeabilization buffer was added to wash the cells three times, followed by washing with PBS three times. Cells were then assessed using Flow cytometry (Agilent, Novocyte 3000). Data analysis was conducted using FlowJo software (version 10.6.2). 2.15 Enzyme-Linked Immunosorbent Assay ELISA was used to measure the secretion of IL-2 in the supernatant of human peripheral CD8 + T cells after treatment with compound 450F10 and stimulation for 24 hours. Human peripheral CD8 + T cells were seeded at a density of 1 × 10 5 cells per well in a 96-well plate and cultured overnight at 37℃ in a 5% CO 2 incubator. The cells were then treated with various concentrations of compound 450F10 (or DMSO) for 30 minutes at 37℃ in a 5% CO 2 incubator. After treatment, 2 μL of human T cell activator CD3/CD28 Dynabeads were added to each well, and the cells were incubated for 24 hours at 37℃ in a 5% CO 2 incubator. After incubation, the cells were centrifuged at 1000 rpm for 3 minutes, and the supernatant was collected. The supernatant was diluted 2-fold and analyzed using an IL-2 ELISA Kit (Absin#abs551102-96T). The absorbance at 450 nm was measured, after subtracting the background reading at 630 nm, to determine the IL-2 secretion levels in the cell supernatant at different compound concentrations. The data were processed and quantitatively analyzed using GraphPad 8.0. 2.16 Statistical Analysis All data were derived from at least three independent experiments with similar results. Data are presented as the mean ± SEM. The data were processed and quantitatively analyzed using GraphPad 8.0. 4. Results 4. 1 High-throughput screening identifies hit Compounds targeting Cbl-b To identify inhibitors targeting Cbl-b, a Cbl-b fluorescence probe was synthesized by attaching a 5-FAM fluorophore to Cbl-b inhibitor compound 54, as described in the patent by Nurix (WO2020264398A1) (Figure S1A). A truncated form of Cbl-b (residues 36–427), containing the TKB domain and the Tyrosine residue at position 363, was expressed and purified. Using the Cbl-b fluorescence probe combined with purified Cbl-b recombinant protein, a high-throughput screening (HTS) method based on fluorescence polarization (FP) was developed to detect compounds’ ability to competitively bind to Cbl-b. ( Figure 2 a). The stability and feasibility of this screening method were validated by testing the reported Cbl-b inhibitor Cbl-b-IN-1 (Figure S1B). Initially, approximately 6000 compounds from an in-house compound library were screened at a single concentration of 50 μM, identifying 54 compounds with over 80% inhibition (Figure S1C). The 54 compounds were subsequently rescreened at a concentration of 10 μM, yielding four candidates (442F9, 450F10, 475H2, and 485E4) that demonstrated over 80% inhibition at this concentration (Figure 2b-c). Further experiments demonstrated that these four compounds competitively bound to Cbl-b in a concentration-dependent manner. Among them, 450F10 exhibited the strongest binding affinity, while 442F9, 475H2, and 485E4 showed slightly weaker binding affinities (Figure 2d). Figure 2. High-throughput screening identifies hit Compounds targeting Cbl-b (a) Flowchart of the high-throughput screening process for hit compounds using fluorescence polarization assay. (b) Inhibition rates of 54 candidate compounds at a single concentration of 10 μM in the initial screening, based on the fluorescence polarization assay. (c) Structures of compounds 442F9, 450F10, 475H2, and 485E4. (d) Binding activity of 442F9, 450F10, 475H2, and485E4, with Cbl-b protein, as determined by fluorescence polarization assays (IC 50 values). All data shown are means ± SEM; n = 3 per group. 4. 2 450F10 directly interacts with Cbl-b protein To validate whether the four hit compounds identified in the screening directly bind to Cbl-b protein, a series of molecular-level experiments were designed and performed. First, the binding of the compounds to Cbl-b was assessed using a protein thermal shift (PTS) assay. The results showed that, except for 485E4, compounds 442F9, 450F10, and 475H2 significantly decreased the thermal stability of Cbl-b, and the negative thermal shift values of the protein increased in a concentration-dependent manner ( Figure 3 a) (Figure S2A). Among them, 450F10 induced the most significant thermal shift. Next, nuclear magnetic resonance (NMR) was conducted to further confirm whether the compounds directly interact with the Cbl-b protein. After incubating Cbl-b with 442F9, 450F10, and 475H2, NMR analysis revealed that only 450F10 directly bound to Cbl-b (Figure 3b). Based on this finding, subsequent experiments focused on 450F10 to further explore its binding mechanism and potential biological significance. To study the binding characteristics of 450F10 to Cbl-b more precisely, surface plasmon resonance (SPR) was employed to measure its binding ability and kinetics. The SPR results demonstrated that 450F10 binds to Cbl-b with a fast association and slow dissociation pattern, showing high affinity, with the binding constant as shown in the Figure 3c-d. Additionally, isothermal titration calorimetry (ITC) was conducted to further confirm the thermodynamic aspects of the direct interaction by measuring heat changes during the binding process. The ITC results revealed that as 450F10 was titrated into the Cbl-b protein, the heat change gradually decreased to a minimum, indicating binding between the two molecules, with the binding constant and stoichiometry (N value) shown in Figure 3e. These findings, from both thermodynamic and kinetic perspectives, confirm the direct interaction between 450F10 and Cbl-b and provide important parameters regarding their binding characteristics. In summary, this study comprehensively utilized various molecular-level experimental techniques based on different detection principles, including FP, PTS, NMR, SPR, and ITC, to qualitatively and quantitatively validate the direct interaction between 450F10 and Cbl-b, providing crucial binding kinetics and thermodynamic parameters. Figure 3. 450F10 directly interacts with Cbl-b protein (a) Melting curves of His-Cbl-b protein (3 μM) treated with 442F9, 450F10, and 475H2. (b) Nuclear magnetic resonance spectra showing direct binding of 450F10 to Cbl-b protein. The CPMG NMR spectra of 450F10 alone (green) and in the presence of Cbl-b protein (5 μM) (red). The STD NMR spectra for 450F10 in the presence and absence of His-Cbl-b protein. (c-d) Surface plasmon resonance measurement of the binding affinity between 450F10 and His-Cbl-b protein. Panel C shows the kinetic plot, and Panel D shows the affinity plot. (e) Isothermal titration calorimetry binding curves for 450F10 (10 μM) titrated with His-Cbl-b protein (100 μM). The upper part of the figure shows the original ITC thermogram, and the lower part shows the fitted binding isotherm curve. 4. 3 450F10 binds to Cbl-b protein through Y260 and F263 To comprehensively reveal the molecular mechanism of the interaction between 450F10 and Cbl-b, and to identify its binding mode and binding sites, molecular docking simulations were performed based on the reported crystal structure of a Cbl-b inhibitor complex (Kimani et al., 2023). The docking results indicated that 450F10 likely binds to Cbl-b at Tyrosine 260 (Y260) and Fnylalanine 263 (F263), preventing phosphorylation of Tyrosine 363 (Y363) by kinases, and thereby inhibiting the activation of Cbl-b protein ( Figure 4 a). To verify this hypothesis, Tyrosine 260 (Y260) was mutated to Fnylalanine, and Fnylalanine 263 (F263) was mutated to Leucine, creating a plasmid for the double mutant Cbl-b (Y260F/F263L) protein. The mutant protein was also expressed and purified from E. coli . A series of molecular-level binding validation experiments were then conducted under the same conditions used for testing the binding between the wild-type protein and 450F10, to assess the interaction between the mutant protein and 450F10. First, the PTS assay showed that 450F10 did not significantly reduce the thermal stability of the Cbl-b (Y260F/F263L) mutant, in contrast to the significant decrease in thermal stability observed with the wild-type Cbl-b protein (Figure 4b-c). Additionally, SPR experiments also supported this conclusion, as no interaction was detected between the Cbl-b (Y260F/F263L) mutant protein and 450F10 (Figure 4d-e). Similarly, ITC experiments with the Cbl-b (Y260F/F263L) mutant protein showed no binding of 450F10 to the mutant protein (Figure 4f). In brief, through molecular docking simulations, the binding sites of 450F10 were predicted, and combined with PTS, FP, SPR, and ITC experiments, the prediction was validated from multiple angles. The results clearly demonstrate that 450F10 binds to Cbl-b protein at Y260 and F263. These findings shed light on the mechanism of action of 450F10 and lay a solid foundation for further structural optimization and the development of more potent Cbl-b-targeted inhibitors. Figure 4. 450F10 binds to Cbl-b protein through Y260 and F263 (a) Molecular docking simulation of 450F10 binding to Cbl-b protein. (b-c) Protein thermal shift binding assay of wild-type Cbl-b protein (3 μM) and Cbl-b (Y260F/F263L) mutant protein (3 μM) with 450F10 (10 μM), under the same experimental conditions as before. (d-e) Surface plasmon resonance assay showing the interaction between His-Cbl-b (Y260F/F263L) mutant protein and 450F10, under the same experimental conditions as before. Panel D shows the kinetic plot, and Panel E shows the affinity plot. (f) Isothermal titration calorimetry binding curves for His-Cbl-b (Y260F/F263L) mutant protein (100 μM) titrated with 450F10 (10 μM). The upper part of the figure shows the original ITC thermogram, and the lower part shows the fitted binding isotherm curve. 4 450F10 inhibits Cbl-b phosphorylation and its E3 ligase-mediated ubiquitination function. To further elucidate the mechanism by which 450F10 inhibits Cbl-b, studies were conducted to verify whether binding to Y260 and F263 could inhibit Cbl-b phosphorylation. As previously mentioned, Cbl-b protein exists in two conformational states: a closed, inactive state and an activated state. In the closed state, the E2 binding site of the RING domain is obscured by the TKB domain. During T cell receptor (TCR) signaling, Src kinase, a key non-receptor Tyrosine kinase, activates Cbl-b by phosphorylating it, thereby triggering its E3 ligase function and contributing to immune negative regulation (Elly et al., 1999). To mimic the phosphorylation activation of Cbl-b in vitro , Src protein was expressed and purified, and an in vitro phosphorylation detection assay using Western Blot to visualize Cbl-b phosphorylation was established. Cbl-b-IN-1 was used to validate the feasibility and reliability of the method (Figure S3A). The in vitro phosphorylation results revealed that 450F10 could concentration-dependently inhibit the phosphorylation of Cbl-b ( Figure 5 a-b). This indicates that 450F10 effectively prevents Cbl-b activation. Upon phosphorylation, activated Cbl-b exhibits E3 ligase activity, where E1 ubiquitin-activating enzyme activates ubiquitin (Ub) and transfers it to E2 ubiquitin-conjugating enzyme. Subsequently, the E3 ligase binds to E2 and the substrate protein, facilitating the transfer of Ub from E2 to the substrate protein, leading to ubiquitination and degradation of the substrate protein. To further confirm the inhibitory effect of 450F10 on Cbl-b E3 ligase function, an in vitro ubiquitination assay system was designed and constructed based on the characteristic autoubiquitination of phosphorylated Cbl-b (Shah, Al-Haidari, Sun, & Kazi, 2021), directly demonstrating Cbl-b’s ubiquitination function. The essential components for constructing the in vitro ubiquitination assay included recombinant purified proteins UBA1 (E1), UbCH5b (E2), and Ub (Gabrielsen et al., 2017) (Dou, Buetow, Sibbet, Cameron, & Huang, 2013). As previously mentioned, these recombinant proteins were purified, and the in vitro ubiquitination assay system was successfully constructed (Figure S3B). The in vitro ubiquitination assay results showed that 450F10 inhibited the self-ubiquitination activity of Cbl-b, in a concentration-dependent manner (Figure 5c-d). At concentrations between 3-6 μM, 450F10 inhibited about 50% of Cbl-b’s ubiquitination activity. To achieve a more precise assessment of activity, an in vitro E2-E3 binding assay was designed and established using homogeneous time-resolved fluorescence (HTRF) to evaluate 450F10’s inhibition of Cbl-b protein functionality in greater detail. This assay measured the interaction between His-tagged UbCH5b (E2) and Flag-tagged Cbl-b (E3), to assess the ubiquitination activity of Cbl-b after activation (Gabrielsen et al., 2017) (Dou et al., 2013). Validation was performed using Cbl-b-IN-1 (Figure S3C). The results of E2-E3 binding HTRF assay revealed that 450F10 inhibited the E2-E3 interaction in a concentration-dependent manner (Figure 5e), indirectly indicating that 450F10 inhibits the autoubiquitination of Cbl-b. To assess whether 450F10 affects Cbl-b’s ability to ubiquitinate downstream substrates, an HTRF-based assay was performed targeting Zap70, a well-known substrate of Cbl-b ubiquitination (Shah et al., 2021). This assay monitored the transfer of GST-tagged Ub to His-tagged Zap70 (Boerth et al., 2023) (Figure S3D). As anticipated, the result shows that 450F10 effectively suppressed Cbl-b-mediated ubiquitination of Zap70, exhibiting inhibitory activity comparable to its ability to inhibit the interaction between Cbl-b (E3) and UbCH5b (E2) (Figure 5f). These findings demonstrate that 450F10 not only inhibits Cbl-b’s autoubiquitination activity but also effectively suppresses its E3 ligase-mediated ubiquitination of downstream substrates. This section demonstrates that 450F10, by binding to Y260 and F263 of Cbl-b, prevents phosphorylation of Y363, inhibits Cbl-b activation, and subsequently suppresses its E3 ligase function. The WB and HTRF experiments collectively confirm that 450F10 not only blocks Cbl-b’s autoubiquitination but also inhibits ubiquitination of downstream substrates. These findings provide critical insights into the inhibitory mechanism of Cbl-b and lay an important foundation for its potential application as a drug target. Figure 5. 450F10 inhibits Cbl-b phosphorylation and its E3 ligase-mediated ubiquitination function. (a-b) In vitro inhibition of Cbl-b phosphorylation experiment. Different concentrations of 450F10 were incubated with recombinant Cblb protein, followed by incubation with purified Src to phosphorylate Cbl-b protein. The samples were then prepared and analyzed by Western blot experiments. Panel B shows the quantitative analysis of the Cbl-b phosphorylation band intensity. All data shown are means ± SEM; n = 3 per group. (c-d) In vitro inhibition of Cbl-b ubiquitination experiment. After incubating the 450F10 with recombinant Cbl-b protein, Src was added to activate the ubiquitin ligase activity of Cbl-b protein. A mixture containing E1 (UBA1), E2 (UbCH5b), and Ub was subsequently added. The samples were prepared and analyzed by Western blot experiments. Panel D shows the quantitative analysis of the Cbl-b ubiquitinated protein band intensity. Saracatinib is a reported Src inhibitor. All data shown are means ± SEM; n = 3 per group. (e) Inhibition of E2 (UbCH5b) and E3 (Cbl-b) interaction by HTRF assay. After incubation with 450F10, Flag-tagged Cbl-b (E3) was incubated with Src, followed by incubation with 6×-His-tagged UbCH5b (E2) and the corresponding fluorescent magnetic beads. The fluorescence intensity was then measured. The DMSO control (dashed line) represents the complete binding activity of Cbl-b with UbCH5b in the absence of the compound (containing only DMSO), and the Reference control (dashed line) represents the background signal in the absence of ATP. (f) Inhibition of Zap70 ubiquitination by HTRF assay. Different concentrations of 450F10 were incubated with Cbl-b (E3), followed by incubation with Src to activate the ubiquitin ligase activity of Cbl-b. A mixture containing His-tagged Zap70, GST-tagged Ub, UBA1 (E1), and UbCH5b (E2) was then added. The fluorescence intensity was measured. The DMSO control (dashed line) represents the complete ubiquitination activity of Cbl-b in the absence of the compound (containing only DMSO), and the Reference control (dashed line) represents the background signal in the absence of ATP. 4.5 450F10 binds to Cbl-b protein intracellularly, modulating downstream pathways and enhancing IL-2 secretion and release in human CD8 + T cells. Building on previous molecular-level findings, 450F10 has been shown to bind to the Cbl-b protein and inhibit its phosphorylation and ubiquitin ligase activity. Cellular thermal shift assay (CETSA) were used to verify the permeability of the compound 450F10 and to determine whether it can bind to the target protein within the cell. The CETSA results demonstrated that, following incubation of Jurkat cells with 450F10, the thermal stability of intracellular Cbl-b protein was significantly reduced ( Figure 6 a-b), indicating that 450F10 also penetrates the cell membrane to interact with full-length Cbl-b inside cells, similarly decreasing its thermal stability. Next, the effects of 450F10 on downstream proteins of Cbl-b were analyzed to assess its inhibitory effect on intracellular Cbl-b. During T cell activation, TCR signaling promotes the phosphorylation of downstream proteins, including PLCγ1, which enhances immune responses (Shah et al., 2021) (Tassi et al., 2005). Cbl-b, as an immune negative regulator in T cells, targets the degradation of p-PLCγ1 via the ubiquitin-proteasome system when activated, thereby suppressing T cell immune function (Q. Liu, Zhou, Langdon, & Zhang, 2014). Therefore, Inhibition of Cbl-b prevents p-PLCγ1 degradation, leading to its accumulation (Boerth et al., 2023) (Mfuh et al., 2024). Based on this principle, Jurkat cells treated with 450F10 and stimulated were collected for Western blot analysis to visualize the phosphorylation level of PLCγ1, indirectly reflecting the inhibitory effect of 450F10 on Cbl-b-mediated cellular ubiquitination. Cbl-b-IN-1 was used as a control to validate the method (Figure S4A-B). The accumulation of p-PLCγ1 in the experimental results indicated that compound 450F10 can inhibit Cbl-b’s ubiquitination activity within cells in a concentration-dependent manner, as shown in the Figure 6c-d. Considering that Jurkat cells are tumor cell lines, which may differ in morphology, function, and signaling pathways from normal human T cells, further evaluation of 450F10’s effect on Cbl-b in human peripheral CD8 + T cells was conducted. Upon activation, T cells release immune factors such as IL-2 (Thien & Langdon, 2001). When the immune negative regulator Cbl-b is inhibited, T cell immune responses are enhanced, resulting in increased IL-2 secretion. Based on this, CD8 + T cells stimulated after treated with different compounds were collected, stained with CD3 and IL-2 antibodies, and analyzed via flow cytometry. The flow cytometry results showed that, compared to the control group, IL-2 secretion levels in CD8 + T cells treated with Cbl-b-IN-1 and 450F10 were upregulated (Figure 6e-f). This indicates that 450F10 can inhibit Cbl-b function in human CD8 + T cells and enhance T cell immune responses. To further quantify the inhibitory effect of 450F10 on Cbl-b in CD8 + T cells, an additional Enzyme-Linked Immunosorbent Assay (ELISA) experiment was performed to measure IL-2 secretion. CD8 + T cells stimulated after treated with the compounds were collected and analyzed for IL-2 secretion in the supernatant using an IL-2 ELISA kit, in accordance with the flow cytometry conditions (Figure S4C). The ELISA data showed that 450F10 can concentration-dependently inhibit Cbl-b activity in human peripheral CD8 + T cells and promote IL-2 release, as shown in the Figure 6g. Additionally, to assess the cytotoxicity of 450F10, a cell proliferation inhibition assay was performed under the same conditions. The results showed that the GI 50 value for 450F10’s inhibition of CD8 + T cell proliferation was six times higher than the EC 50 value for 450F10’s promotion of IL-2 secretion (Figure S4D). This indicates that 450F10 enhances T cell immune function without significantly affecting cell proliferation. Overall, the experimental results from Jurkat cells and human CD8 + T cells indicate that 450F10 can penetrate the cell membrane and bind to the Cbl-b protein at the cellular level. It inhibits the ubiquitination function of Cbl-b, blocks downstream signaling pathways, and promotes the activation of TCR signaling in Jurkat cells. Additionally, Compared to the control group, IL-2 levels in both the intracellular and cell supernatant of CD8 + T cells treated with 450F10 were upregulated, demonstrating it can enhance the secretion and release of IL-2 by human peripheral blood CD8+ T cells. These findings provide experimental evidence for the potential application of 450F10 as an immune modulator. Figure 6. 450F10 binds to Cbl-b protein intracellularly, modulating downstream pathways and enhancing IL-2 secretion and release in human CD8 + T cells. (a-b) WB analysis shows changes in the thermal stability of intracellular Cbl-b protein in Jurkat cells after treatment with 450F10 (10 μM) or DMSO for 1 hour at different temperatures. Panel B shows the grayscale quantification analysis of the thermal stability of intracellular Cbl-b protein after treatment with 450F10. All data shown are means ± SEM; n = 3 per group. (c-d) Dose-response curve of phosphorylated PLCγ1 (p-PLCγ1) levels after exposure to different concentrations of 450F10. The left-most panel shows an unstimulated (Unstim) Jurkat cell as a control, where the total PLCγ1 (TPLCγ1) is completely unphosphorylated. Panel D shows the grayscale quantification analysis of the protein bands from the p-PLCγ1 accumulation experiment. All data shown are means ± SEM; n = 3 per group. (e-f) CD8 + T cells were treated with Cbl-b-IN-1 (5 μM), 450F10 (5 μM), or DMSO for 24 hours, followed by stimulation with CD3/CD28 Dynabeads. The cells were then stained with BV21-CD3 and PE-IL2 antibodies and analyzed by flow cytometry to determine the percentage of double-positive cells in each group. The unstained group and unstimulated group were used as negative controls. The percentage of CD3/IL2 double-positive cells in each group was quantitatively analyzed, as shown in panel F. All data shown are means ± SEM; n = 3 per group. (g) After incubation of CD8 + T cells with 450F10, followed by stimulation with CD3/CD28 Dynabeads for 24 hours, the levels of IL-2 secretion in the CD8 + T cell supernatant were measured by ELISA. All data shown are means ± SEM; n = 3 per group. 5. Discussion Cbl-b is a key negative regulator of immune cells, and extensive research has shown that inhibiting Cbl-b can enhance immune cell responses. In this study, a novel small-molecule Cbl-b inhibitor, 450F10, was successfully identified using a high-throughput screening method based on fluorescence polarization. Its molecular and cellular biological activities were systematically evaluated, and the mechanism of action in inhibiting Cbl-b was explored. The results demonstrated that 450F10 binds stably to key amino acid residues Y260 and F263 in the TKB domain of Cbl-b, preventing phosphorylation of Y363, thereby effectively inhibiting Cbl-b activation and its E3 ligase activity. Furthermore, experimental results showed that 450F10 can penetrate the Jurkat cell membrane, bind to full-length Cbl-b, and inhibit its activity, leading to the accumulation of phosphorylated PLCγ1, a downstream protein in the TCR signaling pathway. Importantly, 450F10 also exhibited immune-enhancing effects in normal human CD8 + T cells by inhibiting Cbl-b function and promoting IL-2 secretion and release, thereby enhancing T cell immune activity. These findings indicate that 450F10 not only inhibits the function of Cbl-b at the molecular level but also enhances immune responses at the cellular level with low toxicity, providing a potential therapeutic window for its application as an immune modulator. Although this study provides strong evidence for the role of 450F10 as a Cbl-b inhibitor, it must be acknowledged that further research is necessary. Future work should include the evaluation of the pharmacokinetic parameters of 450F10 in animal models, as well as its in vivo efficacy and toxicity, to explore its pharmacodynamics and safety. Additionally, considering that the activity of 450F10 as a Cbl-b inhibitor needs to be further enhanced, future studies will focus on the structural modification of 450F10 to improve its binding affinity to Cbl-b and its bioavailability through chemical modifications. Such structural modifications may enhance the activity of 450F10 while reducing its potential side effects. Given the complexity of the immune microenvironment, single-target inhibition may not be sufficient to achieve optimal therapeutic effects. Therefore, exploring combination strategies of 450F10 with other immune modulators or immune checkpoint inhibitors may become a valuable research direction. Combination therapy has the potential to generate synergistic effects, enhancing the immune system’s defense against pathogens while reducing the occurrence of resistance. The development of Cbl-b inhibitors is still evolving, and this study aims to provide valuable insights and ideas for the design of next-generation Cbl-b inhibitors. Author contributions W. H. and Z. F. contributed equally to this work.Conception of the hypothesis: S. Z. and M. Z.; Study supervision: S. Z., M. Z.; Development of methodology: W. H., Z. F.; Acquisition of data: W. H., Z. F., M. H., and M. W.; Analysis and interpretation of data:W. H., Z. F., M. H., M. W., J. Y., Z. G., Y. Z., and J. X.; Writing, review, and/or revision of the manuscript: W. H., Z. F., M. H., M. W., Z. G., and J. X. All authors discuss the study. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Acknowledgements We gratefully acknowledge financial support from National Key Research and Development Program of China (2022YFC3400504 and 2023YFC2305904 to MYZ.), National Natural Science Foundation of China (T2225002 and 82273855 to MYZ, 82474143 to SLZ), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0830200 to MYZ), the Youth Innovation Promotion Association CAS (2023296 to SLZ), the Natural Science Foundation of Shanghai (22ZR1474300 to SLZ), and Young Elite Scientists Sponsorship Program by CAST (2023QNRC001 to SLZ). We thank the staff members of the Large-scale Protein Preparation System (https://cstr.cn/31129.02. 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Keywords drug discovery/target validation immunopharmacology small molecules Authors Affiliations Wei He Nanchang University View all articles by this author Zisheng Fan ShanghaiTech University View all articles by this author Manlin Huang Nanchang University View all articles by this author Min Wu Nanchang University View all articles by this author Zhiming Ge Shanghai Institute of Materia Medica Chinese Academy of Sciences View all articles by this author Jie Yu ShanghaiTech University School of Information Science and Technology View all articles by this author Yuanyang Zhou ShanghaiTech University View all articles by this author Jiahang Xu Beijing Huairou Hospital University of Chinese Academy of Sciences View all articles by this author Mingyue Zheng Shanghai Institute of Materia Medica Chinese Academy of Sciences View all articles by this author Sulin Zhang 0000-0002-9167-4689 [email protected] Shanghai Institute of Materia Medica Chinese Academy of Sciences View all articles by this author Metrics & Citations Metrics Article Usage 355 views 238 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Wei He, Zisheng Fan, Manlin Huang, et al. 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