Design and preliminary efficacy of cp-PCC targeting palmitoylation and expression of PD-L1:
The ZDHHC3 protein is expressed in all breast cancer (BRCA) and HNSCpatients, and in almost all pan-cancer, cervical cancer (CES), and skin cancer and melanoma (SKCM) patients based on immunohistochemistry staining examination results in the Human Protein Atlas database (Fig. 1A). Regardless of the type of cancer, more than half of the patients exhibit high expression levels of the ZDHH3 protein (Fig. 1A). Gene correlations between PD-L1 and ZDHHC3, PD-1, ZDHHC1, and ZDHHC2 in different cancer samples from the TCGA database are shown in Fig. 1B. PD-L1 and ZDHHC3 were significantly and positively correlated in pan-cancer, BRCA, CES, HNSC, and SKCM patients (Fig. 1B).
Western Blot results indicated that downregulation of DHHC1 and DHHC2 did not significantly alter the expression of PD-L1. However, a marked reduction in PD-L1 expression was observed after DHHC3 knockdown (Fig. 1C). These data reveal a robust global association between the key immune checkpoint ligand PD-L1 and the lipid enzyme DHHC3 across cancer types. Based on the aforementioned theoretical framework, we designed and synthesized cp-PCC. The cp-PCC is composed of CPPs, a protein-binding domain peptide sequence, a small-molecule linker, and a small-molecule E3 ubiquitin ligase ligand (Fig. 1D). We employed the TAT sequence as the CPP, linking it to a protein-binding domain sequence that has been previously validated for the effective targeting of DHHC3(43). The assembly was connected via a polyethylene glycol linker. In this study, human breast cancer cells (MDA-MB-231), human cervical cancer cells (C33A), human head and neck squamous carcinoma cells (FaDu), and human malignant melanoma cells (A375) were treated with 10 µM of PCC series drugs. Following a 6-h incubation period, Western Blot analysis was conducted to assess the levels of PD-L1 protein after treatment. The results indicated that, compared to the control group, the degradation of the PD-L1 protein was observed in all four cell lines treated with PCC16/17/18 (Fig. 1E).
cp-PCCs significantly decrease PD-L1 protein in cell lines:
We tested three small-molecule E3 ubiquitin ligase ligands: CRBN, VHL, and IAP. The cp-PCCs linked to these three ligands were designated as PCC16, PCC17, and PCC18, respectively. To investigate the degradation levels of DHHC3 and PD-L1 proteins by the PCC16/17/18 series of drugs, we initially treated C33A cells with the drugs at concentrations of 0.1/0.5/5/10/50 µM. Subsequently, we used Western Blot analysis to detect the levels of DHHC3 and PD-L1 proteins following treatment with the cp-PCC series of drugs. The Western Blot results indicated significant degradation of DHHC3 and PD-L1 proteins at 0.1 µM by PCC16 (Fig. 2A), at 5 µM by PCC17 (Fig. 2B), and at 10 µM by PCC18 (Fig. 2C). Additionally, we calculated the DC50 values for the three drugs, with CRBN-based PCC16 exhibiting the best degradation activity among the three (DC50 = 0.103 µM), VHL-based PCC17 with a DC50 of 1.92 2µM, and IAP-based PCC18 with a DC50 of 7.530 µM (Fig. 2D). Additionally, we incubated the cp-PCC series of drugs with C33A cells at various time points (0, 1, 2, 4, 8, 12, and 24 h). The analysis indicated that a minimum incubation period of 4 h was necessary to achieve significant degradation of the target proteins DHHC3 and PD-L1 (P < 0.05) (Fig. 2E-G). Using confocal microscopy, we dynamically monitored drug penetration into the cell membrane at different time intervals. The results revealed a gradual increase in the intracellular accumulation of PCC16, PCC17, and PCC18 with extended incubation time (Fig. 2H). Additionally, in murine cervical cancer cells (U14), colon cancer cells (CT26), and melanoma cells (B16F10), Western Blot analyses were conducted to evaluate the levels of PD-L1 protein after treatment with PCC16 at varying concentrations (0.1, 0.5, 5, 10, and 50 µM). The results indicated a concentration-dependent decrease in PD-L1 protein levels across all cell types (Fig. S1). Collectively, these findings demonstrate that the degradation of the target protein DHHC3 and the decrease in PD-L1 induced by the PCC16/17/18 drugs were both time- and concentration-dependent.
Synthesis and validation of the cp-PCC pharmaceutical compound:
The structural configuration of the cp-PCC compound is illustrated in Fig. 3A and Fig. S2), which is consistent with the rhodamine and TAT groups. The synthesized peptides were purified by HPLC to achieve a purity level exceeding 95%. The synthesis and verification of the compound were performed by the Chinese Peptide Company (Hangzhou, China), and the synthetic route was shown in Figure. 3B. Throughout the purification phase, HPLC was performed for Quality Control (QC). Subsequent to the attainment of QC clearance, the sample underwent lyophilization to yield a solid powder form (Fig. 3C-E, Fig. S3).
PCC16 exhibited stable binding affinity to the target protein DHHC-3:
In this study, after incubating C33A cells with 2.5 µM of PCC16 for 6 h, high-resolution confocal microscopy revealed the colocalization of the DHHC3 protein and PCC16 (Fig. 4A), suggesting potential effective binding between the drug and the target protein DHHC-3. Further analysis of the target specificity of PCC16 in C33A cells was conducted using the cellular thermal shift assay. Cells, with or without PCC16 treatment, were heated at 37–61°C for a specific duration, followed by Western Blot analysis. The results indicated that, in the untreated group, significant degradation of DHHC3 occurred at 49°C; however, in the PCC16-treated cells, DHHC-3 was largely degraded only at 55°C (Fig. 4B). The temperature-protein expression curves plotted from these data showed a slower decline in DHHC3 protein levels with increasing temperature in the treated group, with a noticeable right shift in the melting curve (Fig. 4C). These results suggest that PCC16 effectively binds to DHHC3, thereby altering its thermal stability. Additionally, the cellular uptake of the drug by C33A cells was quantified using flow cytometry to measure the fluorescence intensity. As the treatment duration and drug concentration increased, a gradual increase in cells exhibiting red fluorescence was observed, indicating the presence of PCC16 (Fig. 4D). To assess the cytotoxic effects of PCC16 on C33A cells, the MTT assay was conducted to monitor the changes in cell numbers across different times and concentration gradients. Even after incubating the cells for up to 24 h or at concentrations as high as 50 µM, the drugs exhibited relatively low cytotoxicity with no significant differences between groups (Fig. 4E).
Investigation of the degradation mechanism of cp-PCC compounds:
We further explored whether the cp-PCC series of drugs enhance the ubiquitination of DHHC-3 via the proteasome pathway, thereby promoting the degradation of PD-L1. Western Blot results showed that after 6 h of treatment with PCC16/17/18, the protein levels of DHHC-3 and PD-L1 decreased. Addition of the proteasome inhibitor MG132 significantly reduced the degradation of DHHC-3 and PD-L1 proteins (Fig. 5A-B). Concurrently, immunofluorescence results revealed that the fluorescence intensity of PD-L1 decreased after the addition of PCC16/17/18 and was significantly reversed upon the addition of MG132 (Fig. 5C). These results suggest that PCC16/17/18 can enter cells and inhibit the PD-L1 protein through the proteasome pathway.
Decrease of PD-L1 via DHHC3 induced by cp-PCCs enhanced chemotherapeutic sensitivity and anti-tumor immunity:
To compare the inhibitory effects of PD-L1 monoclonal antibody and PCC16/17/18 on PD-L1 protein, Western Blot results indicated that at the same concentration, the degradation effect of PCC16/17/18 on C33A cells was significantly superior to that of the PD-L1 monoclonal antibody (Fig. 6A). To investigate whether PCC16 increases the sensitivity of C33A cells to cisplatin, we evaluated the combined effects of cisplatin and PCC16 using TUNEL and Ki67 assays. The results showed that, compared to the control group, cell proliferation decreased and apoptosis increased in the cisplatin group; compared to the cisplatin group alone, the combination of PCC16 and cisplatin significantly reduced proliferation and increased apoptosis, with statistically significant differences (Fig. 6B-C). The colony formation assay, as shown in Fig. 6D, indicated that compared to the control group, the number of C33A colonies decreased in the cisplatin group and significantly decreased in the PCC16 plus cisplatin group, suggesting that PCC16 can enhance the proliferation-inhibitory effects of cisplatin on the C33A cell line. We constructed a T-cell-C33A co-culture system, incubated C33A cells with different concentrations of PCC16 for 6 h, and measured cytokine levels in the supernatant using ELISA. Compared to the control group, PCC16-treated cells significantly increased the secretion of IFN-γ and TNF-α (P<0.05) (Fig. 6E). In the co-culture system, after incubation with different concentrations of PCC16 for 6 h, Hoechst 33528 staining was performed to detect the fluorescence intensity of the tumor cells. PCC16 enhanced the T-cell-mediated killing of C33A cells in a concentration-dependent manner (Fig. 6F). These results suggest that PCC16 degrades PD-L1, thereby blocking the interaction between PD-L1 and PD-1, promoting the secretion of IFN-γ and TNF-α into the supernatant of the co-culture system, and ultimately increasing tumor cell apoptosis within the system.
PCC16 demonstrated efficacious in vitro and in vivo therapeutic effects in combating ICB-resistant tumor:
The 4T1 cell line is a breast cancer cell line derived from mouse mammary tumor tissue and has been demonstrated to be resistant to ICB drugs. To validate the efficacy of cp-PCC drugs in ICB-resistant tumors, we assessed the degradation effect of PCC16 on cellular PD-L1 protein in 4T1 cells using Western Blot analysis after treating the cells with concentration and time gradients. The results demonstrated a significant degradation in PD-L1 protein levels in 4T1 cells under the treatment of PCC16, with substantial statistical significance (P < 0.05) (Fig. S4). Subsequently, 4T1 cells were injected into the dorsum of BALB/c mice to establish a 4T1 cell xenograft mouse model and study the in vivo therapeutic effects of PCC16. When the tumor volume reached 80–100 mm3, the mice were randomly divided into four treatment groups (n = 4 each): saline (control), PD-L1 inhibitor, BMS-8, PD-L1 monoclonal antibody, and PCC16 (Fig. 7A). After 21 days of treatment, tumor growth curves were calculated and plotted based on tumor appearance (Fig. 7B) and growth (Fig. 7C). In the control group, the tumor tissues exhibited the fastest growth and largest volume. Compared to that in the PD-L1 monoclonal antibody and small-molecule PD-L1 inhibitor (BMS-8) groups, PCC16 significantly inhibited tumor growth. Body weight changes in the four groups of mice were not significantly different (Fig. 7D). Tumor weights were measured in each group, and the control group had the largest tumors, whereas the average tumor weights in the BMS-8, PD-L1 monoclonal antibody, and PCC16 groups were 90.8%, 77.9%, and 10.1% of that in the control group, respectively. Compared with the control group, the tumors in the BMS-8 and PD-L1 treatment groups showed a slight decrease in weight and size. However, in the PCC16 treatment group, there was a significant decline in both tumor weight and size compared to the control group, as well as the BMS-8 and PD-L1 monoclonal antibody treatment groups (P < 0.05) (Fig. 7C, E). TUNEL staining was used to assess the impact of PCC16 on apoptosis in mouse tumor tissues; compared to the control group, the number of apoptotic tumor cells significantly increased in the PCC16 treatment group (Fig. 7F). Post-PCC16 treatment resulted in notable inhibition of tumor cell proliferation, as demonstrated by anti-ki67 staining (Fig. 7G). Western Blot and immunohistochemistry analyses of randomly collected tumor samples from the PBS and PCC16 groups (Fig. 7H, I) indicated that PCC16 induced PD-L1 protein degradation in vivo. Additionally, histological hematoxylin and eosin staining of the heart, liver, spleen, lungs, and kidneys of mice showed no signs of toxicity (Fig. 7J), indicating good tolerance in all mouse groups throughout the in vivo experiments. These results suggested that PCC16 exhibits strong antitumor activity in vivo.