PMCID string | Title string | Sentences string |
|---|---|---|
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Copy numbers of JEV RNA were normalised to GAPDH levels using comparative cycle threshold values (2) determined in parallel. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The following primer sets were used for amplification. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Specifically, the primers were designed against the 3′ untranslated region of the JEV SA14 strain (GenBank accession number: U14163.1). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | JEV: forward 5′-CCCTCAGAACCGTCTCGGAA-3′, reverse 5′-CTATTCCCAGGTGTCAATATGCTGT-3′; GAPDH: forward 5′-TGGGCTACACTGAGCACCAG-3′, reverse 5′-AAGTGGTCGTTGAGGGCAAT-3′. The assay was conducted as previously described . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Following collection in RIPA buffer (Thermo Fisher Scientific), cells were lysed by sonication and denatured by boiling. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Protein samples were normalized by concentration, resolved by 10% SDS-PAGE, and electrotransferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Subsequently, the membranes were blocked with 5% skimmed milk for 2 h at room temperature prior to incubation with the specified primary and HRP-conjugated secondary antibodies. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Protein bands were visualised with the ECL Plus enhanced chemiluminescence detection reagents (PerkinElmer Life Sciences, Waltham, MA, USA), depending on signal intensity. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The assay was conducted as previously described . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | At 48 h post-transfection with the indicated plasmids, cells were harvested from 6-well plates. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Cells were lysed on ice for 4 h using a lysis buffer composed of 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 10 µg/mL leupeptin, 10 µg/mL aprotinin, and 2 mM PMSF. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Following centrifugation of cell lysates at 13,000 rpm for 10 min, the resulting supernatants were incubated for 1 h with 1 µg of either anti-GSPT1 antibody or a rabbit IgG isotype control (GeneTex, GTX213110-01). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Subsequently, 20 µL of protein G agarose beads (Roche, Basel, Switzerland) were added, and the mixture was incubated overnight at 4 °C with gentle rotation. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | After centrifugation, pellets were washed with lysis buffer, and target proteins were detected by Western blotting using specific antibodies. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | To elucidate the stage of the viral life cycle targeted by CC-90009, JEV-infected SH-SY5Y cells (MOI = 0.5) were treated with 10 µM compound at various time points relative to infection, ranging from 4 h pre-infection to 24 h post-infection at 4 h intervals. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | All infections and treatments were carried out at 37 °C. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Viral infection levels were quantified by IF. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | One-day-old Institute of Cancer research suckling mice were subcutaneously inoculated with JEV at a dose of 4 × 10 plaque-forming units per mouse. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Mice were subsequently administered CC-90009 (20 mg/kg) subcutaneously once daily for five consecutive days. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The compound was formulated in a vehicle containing dimethyl sulfoxide (DMSO) and 20% sulfobutylether-β-cyclodextrin (HY-17031, MedChemExpress) in saline (10:90, v/v). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Control-infected mice received the same volume of DMSO and 20% sulfobutylether-β-cyclodextrin. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The survival rates were assessed daily until 14 days post-infection. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Organs and tissues were collected from euthanised mice at the indicated time points and fixed with 4% paraformaldehyde (Servicebio, Wuhan, China) at 4 °C for 24 h. After fixation, tissues were subjected to histopathological and immunohistochemical analyses. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | All data represent the arithmetic mean ± standard deviation (SD) of a minimum of three independent replicates. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Two-group comparisons were assessed by Student’s t-test, while differences across multiple groups were evaluated using one-way ANOVA with Bonferroni’s correction for post hoc testing, implemented in GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | A p-value < 0.05 was considered statistically significant. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | To investigate the antiviral activity of TPD-based compounds against JEV infection in SH-SY5Y cells, we selected five clinically advanced compounds: CC-90009; CC-92480, a phase III anti-myeloma CELMoD; NVP-DKY709, a phase I anti-tumour CELMoD targeting Ikaros family zinc finger protein 2; ARV-110, a phase II androgen receptor targeting PROTAC; and ARV-471, a phase II oestrogen receptor PROTAC for breast cancer. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | SH-SY5Y cells were co-treated with JEV and each compound, followed by assessment of their effects on viral infectivity. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Screening experiments revealed that CC-90009 significantly inhibited JEV infection in SH-SY5Y cells, whereas the other compounds showed negligible anti-JEV activity (Figure 1A). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Further investigations across a broad concentration range confirmed CC-90009′s anti-JEV activity and cytotoxicity. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | As shown in Figure 1B–D, it exerted a dose-dependent inhibitory effect on JEV infection with a 50% maximal inhibitory concentration (IC50) of 3.847 μM and a 50% cytotoxic concentration (CC50) of 333.9 μM, highlighting its potential as a promising antiviral agent. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | To identify the stage of the JEV lifecycle inhibited by CC-90009, a time-of-addition assay was performed in which the compound was administered to infected SH-SY5Y cells at various times relative to infection. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | As shown in Figure 2A,B, compared with CQ, a well-known viral entry inhibitor, CC-90009 exhibited significant suppressive activity across all therapeutic windows except pretreatment, suggesting that CC-90009 might affect both viral entry and post-entry stages. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | JEV entry mainly comprises binding and endocytosis. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | We initially examined the role of CC-90009 in the viral binding process. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | SH-SY5Y cells were co-incubated with JEV and CC-90009 at the indicated concentrations for 1.5 h on ice. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The results indicated that CC-90009 did not affect JEV infectivity during the binding stage in SH-SY5Y cells (Figure 2C). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | In contrast, treatment with the positive control inhibitor heparin resulted in a significant reduction in viral infection. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | We next investigated whether CC-90009 influences viral endocytosis. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | It is well established that JEV employs a caveolin-dependent pathway for entry into neuronal cells . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Given this established mechanism, we employed RT-qPCR to assess whether CC-90009 impairs JEV entry via this route. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Following a 1.5 h incubation of SH-SY5Y cells with the virus on ice, the supernatant was replaced with fresh medium containing CC-90009 prior to a further 1 h incubation at 37 °C. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | After treatment with proteinase K to remove uninternalised virus, intracellular JEV was quantified. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Unlike the positive control nystatin, CC-90009 did not reduce JEV RNA levels, indicating that CC-90009 does not affect viral endocytosis (Figure 2D). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The above-mentioned results demonstrated that CC-90009 does not affect JEV entry. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | We therefore investigated whether CC-90009 impacts post-entry stages of JEV infection. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Following endocytosis, JEV first translates viral non-structural proteins, which then assemble into replication complexes on the endoplasmic reticulum to facilitate viral RNA replication. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | To determine if CC-90009 affects JEV translation and replication, SH-SY5Y cells were treated with CC-90009 immediately following JEV infection to evaluate its effects on viral RNA accumulation and non-structural protein expression during a 24 h time course. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | At 6 h post-infection, CC-90009 mildly decreased JEV RNA accumulation. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | However, from 12 to 24 h post-infection, CC-90009 significantly reduced JEV RNA accumulation (Figure 3A). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | CC-90009 also significantly diminished the accumulation of GSPT1 and JEV non-structural proteins compared to the DMSO-treated control at 24 h post-infection. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Notably, the inhibitory effect of CC-90009 on NS5 protein levels was the most prominent (Figure 3B). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | CC-90009 functions as an E3 ubiquitin ligase modulator by selectively recruiting GSPT1 to the CRL4–CRBN complex, leading to its targeted ubiquitination and subsequent degradation. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | To elucidate the role of GSPT1 in JEV replication and translation, siRNA-mediated knockdown (KD) was employed to assess its impact on both viral RNA accumulation and non-structural proteins expression. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Compared with the control group, GSPT1-KD did not significantly suppress JEV RNA levels (Figure 4A), but led to a reduction in the abundance of JEV non-structural proteins (Figure 4B). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | This finding aligns with the established role of GSPT1 as an essential constituent of the eukaryotic translation termination complex (eukaryotic release factor 1/eukaryotic release factor 3) . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Given the subtle impact on JEV RNA accumulation but a decrease in JEV protein levels, we reason that GSPT1 may support JEV infection by facilitating viral protein translation. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | In contrast to siRNA-mediated GSPT1 depletion, treatment with CC-90009 suppressed the accumulation of JEV RNA following infection, indicating a fundamental defect in JEV RNA synthesis. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | We initially investigated a potential physical interaction between GSPT1 and the JEV NS5 protein using co-IP. ( |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Figure 5A). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Furthermore, treatment with CC-90009 significantly enhanced the ubiquitination of GSPT1, whereas the ubiquitination level of JEV NS5 was unaffected (Figure 5B). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Collectively, we hypothesize that CC-90009-mediated tethering of the E3 ligase to GSPT1 may lead to the concomitant degradation of the bound JEV NS5 protein. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | To validate this hypothesis, cells overexpressing GSPT1 and NS5 were treated with either CC-90009 or the proteasome inhibitor MG132, and subsequent alterations in protein levels were examined. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Treatment with CC-90009 alone reduced the levels of both GSPT1 and NS5, whereas MG132 alone had no significant effect. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | However, co-treatment with MG132 abrogated the degradation induced by CC-90009, restoring protein levels to those observed with MG132 treatment alone (Figure 5C). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Furthermore, CRBN-KD attenuated the CC-90009-induced degradation of both GSPT1 and JEV NS5 (Figure 5D). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | These results suggest that the degradation of both GSPT1 and NS5 following CC-90009 treatment is mediated by the proteasome pathway. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Consequently, diminished levels of JEV NS5 are likely to result in a global suppression of viral RNA synthesis. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Collectively, our findings demonstrate that CC-90009 exhibits potent anti-JEV activity in SH-SY5Y cells. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | We next extended our analysis to determine whether the antiviral activity of CC-90009 extends to other mosquito-borne viruses. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | As shown in Figure 6A, CC-90009 treatment potently suppressed infections with both CHIKV and WNV in SH-SY5Y cells in a dose-dependent manner. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Based on the IC50 values, CC-90009 exhibited the strongest antiviral activity against CHIKV (IC50 = 10.6 μM), followed by WNV (IC50 = 23.16 μM). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Furthermore, time-window assays revealed that CC-90009 predominantly targets post-entry stages of both WNV and CHIKV infection (Figure 6B). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Co-IP assays confirmed that GSPT1 physically interacts with NS5 of WNV and nsP4 of CHIKV (Figure 6C). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | To evaluate the in vivo antiviral efficacy of CC-90009, we examined its protective effect against JEV infection in a murine model. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Suckling Institute of Cancer Research mice were inoculated subcutaneously with a lethal dose of JEV and subsequently administered either DMSO or CC-90009. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Survival was monitored daily. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Mice in the JEV-infected, DMSO-treated group began exhibiting clinical signs—such as generalised tremors and unilateral limb paralysis—at three days post-infection. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Mortality reached 83.3% (10/12) between four and seven days post-infection (Figure 7A). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | In contrast, CC-90009 treatment significantly alleviated symptoms and reduced the mortality rate to 38.5% (5/13). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Mice subjected to repeated CC-90009 treatment exhibited a markedly lower viral load in brain tissue than those in the DMSO-treated control group. ( |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Figure 7B). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Further analysis using hematoxylin and eosin (H&E) staining revealed that the CC-90009-treated group exhibited less severe histopathological damage in brain tissues compared with JEV-infected controls (Figure 7C). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Additionally, immunohistochemical examination of mouse brain tissue showed high abundance of viral particles in the control group, whereas CC-90009 administration notably reduced the number of viral particles (Figure 7D). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | In this study, we found that CC-90009 acts as a broad-spectrum antiviral agent, inhibiting infections by JEV, WNV, and CHIKV in SH-SY5Y cells. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Mechanistically, CC-90009 suppresses JEV translation processes and impairs viral genome replication by interfering with the protein levels of JEV NS5. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | In vivo experiments demonstrated that CC-90009 significantly protects suckling mice from JEV infection-induced mortality. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Here, we report for the first time that CC-90009 treatment induces the degradation of both GSPT1 and JEV NS5. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The NS5 protein is the largest and most conserved enzyme encoded by flaviviruses. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | It is characterized by two functional domains: an N-terminal methyltransferase and a C-terminal RdRp . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The RdRps of flaviviruses are structurally similar: JEV and WNV share 70% identity with Zika virus, while Dengue virus (DENV)-2 and DENV-3 share 76% and 81% identity with Zika virus, respectively . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Based on the conservation of NS5 and its similarity between JEV and WNV, CC-90009 effectively inhibits both flaviviruses, most likely by targeting the NS5 protein of each virus. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | In the case of CHIKV, a member of the Alphavirus genus, its nsP4 protein also exhibits RdRp activity ; however, the precise mechanism of action of CC-90009, including its potential targeting of nsP4, remains to be elucidated. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Collectively, these findings suggest the potential for developing CC-90009-based therapeutics against JE by targeting the JEV RdRp for degradation. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Our results demonstrated that CC-90009 treatment inhibited JEV infection in neuronal cells, with a significant reduction in NS5 protein levels. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | When proteasome function was inhibited by MG132, NS5 degradation was abrogated. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Furthermore, CC-90009 failed to degrade NS5 upon CRBN inhibition. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | These findings collectively indicate that CC-90009 exerts its function by targeting NS5 for degradation via the proteasome pathway. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | However, we did not detect ubiquitination of NS5, suggesting that NS5 degradation may not occur through direct ubiquitination; instead, it might be indirectly targeted for proteasomal degradation together with GSPT1. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | By reprogramming the substrate specificity of the CRL4–CRBN E3 ligase, CC-90009 may convert JEV NS5 into a neosubstrate, thereby targeting it for degradation. |
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