Cervical cancer is the second most widespread malignant tumor in women, with more than 500,000 new cases and 300,000 deaths annually worldwide (1–3). Patients diagnosed with early-stage cervical cancer remains a curable disease and are expected to have excellent survival outcomes, but most advanced or recurrent cervical cancer casesprognosis have rather disma prognoses (4). Effective treatments available for advanced cervical cancer are limited and are currently restricted to radiotherapy supplemented with chemotherapy (5–8). In the past several years, tumor-immunotherapy has emerged as a key candidate therapy. The cancer checkpoint blockade with inhibitors that target the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway have demonstrated promising results for a wide variety of tumors (9, 10). Some PD-1/PD-L1 inhibitors have been evaluated for their effects on cervical cancer in clinical trials, revealing a degree of clinical benefits. Among these, a monotherapy using the PD-1 inhibitor pembrolizumab has been approved by the U.S. Food and Drug Administration (FDA) to treat patients with persistent, recurrent, or metastatic cervical cancer with positive PD-L1 expression, which provides hope for future clinical interventions (11, 12). However, as a single agent, pembrolizumab has limited efficacy in treating cervical cancer and should be combined with other treatments. One clinical study reported that the response rate to anti-PD-L1 treatment in patients with PD-L1 positive tumors was less than one fifth (13, 14). Furthermore, primary and acquired drug resistance gradually reduce the efficiency of immune checkpoint inhibitors (ICIs) treatment, especially in cervical cancer (15). Multiple mechanisms would result to the drug-resistance to ICIs, but PD-L1 overexpression is a common outcome in this situation (16, 17).

To improve its efficiency and overcome potential drug resistance, targeted protein degradation has been evaluated as a potential strategy (18). Proteolysis-targeting chimeras (PROTACs) have been applied in cancer treatment strategies for several years, and many of these drugs manufactured have entered the clinical experimentation stage (19). However, the limitation of this tech so far is linking small molecular drug (20). The reason for that is targeting human protein is challenging due to the difficulty of identifying kinase or protein binding pocket structure that bind small molecules. Thus, around 80% of proteins are currently considered “untargetable” or “undruggable” (21–23).Protein–protein interaction (PPI) has been easier to integrate into protein or peptide drug development, especially for intracellular targets (24). Compared to small molecules inhibitors, PROTACs bring the degradation function to the target, negating the need for an active site and expands the accessible targets well beyond those that are druggable by traditional stoichiometric inhibition (25–28). Meanwhile, peptide drugs are less toxic than small molecules inhibitors (29–31).Stapled peptides are a modified peptides by a covalent bonds to form a local alpha-helical structure. Compared to linear peptides, stapled peptides can yield improved stability, target affinity, and ability to cross the cell membrane (32–34). In our previous studies, we designed peptide-based PROTACs to decrease PD-1/PD-L1 expression (35). However, we found that targeting DHHC3, the acyltransferase of palmitoylation of PD-L1, yielded a significantly better effect than that obtained by directly targeting PD-L1 in human cancer cells. Here, we synthesized and optimized a stapled peptide to bind DHHC3 and compared its effects on decreasing PD-L1 expression in human cervical cancer cells in contrast to those of the FDA approved PD-L1 inhibitor.

We treated cervical cancer cell lines C33A and HeLa with the designed stapled peptide for 4 h and observed strong degradation activity against PD-L1. Moreover, we found that treatment downregulated the target protein to < 50% of baseline level at 0.1 μM. PD-L1 degradation occurred rapidly, with significant degradation occurring after 4 h of treatment with 250 nM peptide ( Figure 2 ). Palmitoyltransferase ZDHHC3 (DHHC3) as the main acyltransferase required for the palmitoylation of PD-L1. When C33A was treated with the stapled peptide in the same dose and time-dependent manners, the expression of DHHC3 was gradually decreased ( Figure 3A ). To determine if the stapled peptide was able to engage with DHHC3 in the complex cellular milieu across a range of temperatures (37–61°C), lysate from C33A cells was incubated with peptide at a single compound concentration (0.1 μM). The results indicated that the peptide promoted DHHC3 stabilization at the concentrations examined ( Figure 3B ).

Flow cytometry was used to measure fluorescence intensity, which revealed that degradation changed in a time- and dose-dependent manner ( Figure 4A ). We used electronic and confocal laser microscopy to assess whether the peptides penetrated the cell membrane, and the results demonstrated that the peptide could completely cross the cell membrane after 4 h of incubation ( Figure 4B ). In a separate experiment, we found that the proteasome inhibitor MG132 could alleviate the peptide-mediated degradation of PD-L1 in C33A and HeLa ( Figure 5A ). Moreover, the fluorescence intensity of PD-L1oberserved in cells through electronic and confocal laser microscopy was rescued with MG132 treatment, and the fluorescence intensity tendency remained corresponding with that of PD-L1 tested by western blotting ( Figure 5B ). Therefore, the data demonstrated that the stapled peptide degraded PD-L1 in a proteasome-dependent manner.

Because the PD-1/PD-L1 pathway is closely connected to T cell proliferation and inflammatory cytokine expression, we established a T cell–C33A cell co-culture assay to evaluate the effects of the stapled peptide. Treatment with the peptide induced IFN-γ and TNF-α release in a dose-dependent manner by degrading PD-L1, with the effects being more significant than those resulting from BMS-8 treatment ( Figure 6A ). Hoechst 33528 staining was used to measure fluorescence intensity, revealing that the stapled peptide enhanced T cell killing ability of C33A in a concentration-dependent manner after co-culture ( Figure 6B ).

To further confirm the effects of stapled peptide on PD-L1 degradation, we compared the PD-L1 inhibitor BMS-8 and our stapled peptide in terms of PD-L1 degradation ability in C33A and HeLa cells. Cells were treated with 0.1 μM peptide or BMS-8 induced within 4 h, the results showed the stapled peptide decreased PD-L1 more effectively than BMS-8 ( Figure 7A ). Western blotting further revealed that the stapled peptide induced PD-L1 degradation with a DC50 = 0.054 μM, while BMS-8 induced degradation with a IC50 = 7.789 μM, in C33A cells. Similarly, in HeLa cells, the stapled peptide showed a significant decrease of PD-L1 (DC50 = 0.044 μM) compared with BMS-8 the stapled peptide (IC50 = 7.485 μM). These results suggest that the stapled peptide exerted a striking downregulation effect on PD-L1 ( Figure 7B ).

The stapled peptide was also tested for cytotoxicity by the MTT assay in C33A and HeLa cells. Both cell lines were exposed to increasing concentrations of the peptide for 4 h or treated with 0.1 μM for increasing periods. Our results demonstrated that the SP-PROTAC had low toxicity in C33A and HeLa cells up to a 24 h incubation time and dose of 2.5 μM ( Figure 8 ).

PROTACs are designed to utilize intracellular bioactivity mechanisms and difficult to target for the checkpoint proteins (18–20). In a previous study, we evaluated the ability of a peptide-PROTAC to degrade PD-1 and PD-L1 proteins in human cancer cells, which exhibited potent function at micro-mol/L concentrations (34). However, linear peptide–based drugs are generally unstable under in vivo or plasma conditions, and peptide degradation significantly limits their applicability (33). Stapled peptide modification is considered to be useful for achieving a stable and potent mini-protein structure–like binding effect with targets. The stapled peptide can be connected with an amino acid peptide linker and binder to E3 ubiquitin ligases, forming a new staple peptide–based PROTAC compound (37).

In the present study, we first synthesized a degrading compound with a stapled structure based on linear peptide binding of DHHC3 with the aim of degrading PD-L1 in human cervical cancer; the stapled peptide exhibited a potent inhibition effect. we treated human cervical cancer cells lines C33A and HeLa to asssess whether the SP-PROTAC has a more stable structure. Our results show that the modified peptide drug effectively penetrated the cell membrane. Penetration ability is one primary issue limiting the utility of peptide-based drugs as they cannot easily enter cells due to their relatively large molecular weights (26). Recently reported cell penetrating peptides (CPPs) have addressed this obstacle to some extent. The trans-activator protein Tat was first identified the function of cell membrane penetrating in HIV-1 transcription (38). Since then, numerous CPPs have been confirmed and applied in peptide-based drug development (39–41). However, there are some limitations regarding the application of CPPS: firstly, the low specificity of CPPs may result in toxicity to human cells; secondly, CPPs are relatively unstable in vivo before the drug arrives to the target (21). These shortcomings make them unfavorable for the clinical translation of peptide-based drugs. In contrast to CPPs, stapled peptides can better penetrate cell membranes by forming a relative stereo structure modified by the substitution or change of the net charge in the designed peptides. Moreover, the modified structure can also hide the restriction sites of linear peptide to promote stability (35). In this study, we observed a potent and stable inhibition effect as our synthesized SP-PROTAC compound effectively degraded PD-L1 at a dose of 25 nmol. Furthermore, to confirm that our observations were attributed to proteolysis degradation, we blocked the E3 ubiquitin pathway by MG132, and the decrease in PD-L1 recovered correspondingly.

Thus, we verified that our SP-PROTAC effectively limited PD-L1 in human cervical cancer cells. Because stapled peptides are a relatively new class of synthetic compounds, we tested whether the peptide itself conferred any toxicity to human cells. The MTT assay was performed to evaluate cell viability after treatment. We found that the SP-PROTAC had a low toxicity to C33A cells up to a 24 h incubation time and a dose of 50 μM. The compounds still showed significantly higher inhibition of cell-intrinsic PD-1/PD-L1 interaction in T cells and beneficial effects on T cell function. Additionally, and treatment with SP-PROTAC induced TNF-α and IFN-γ release in a dose-dependent manner, which confirmed the function of our SP-PROTAC.

Although several small molecule inhibitors have been identified to affect the PD/PD-L1 pathway, small molecules remain high challenging to utilize in drug development (21). Immobilization in the design process of SP-PROTAC improves controllability and subsequent applicability. We used an amino acid linker to connect with the binder of von Hippel-Lindau, a high-efficiency E3 ligase, which induced potent degradation for targeting proteins (18). Small molecules may be more advantageous for diversifying the selection of E3 ligases, but they are limited to relatively few binding structures in most proteins (21), which hinders their targeting ability. In our previous study, an appropriate binding peptide sequence for targeting DHHC3 was determined through PPI by a program utilizing artificial intelligence (34). Bristol-Myers Squibb (BMS) has disclosed the patent claim with structures of a number of BMS compounds, which are the potential inhibitors of the PD-1/PD-L1 pathway. One of the BMS compounds, BMS-8, binds to PD-L1 directly and induces formation of PD-L1 homodimers, which in turn prevents PD-1 interaction in previous studies (42, 43). Here, we optimized the sequence to synthesize the SP-PROTAC. The effect of our synthesized SP-PROTAC compound was much better than the conventional small molecule inhibitor of PD-L1, BMS-8 as the control to compare its effect with SP-PROTAC.

There are three main mechanisms of small molecule inhibition of PD-L1 function: blocking reaction of PD-1 to SHP2, inducing the autophagic reaction of PD-L1, and blocking PDL1/PD-1 interaction (44–47). BMS-8 can strongly bind to the monomer molecule of PD-L1 and result in homodimer formation, which inhibits the interaction between PD-1 and PD-L1 (48, 49). With a molecular weight of only 494.4, BMS-8 can work effectively on a low micro-mol/L IC50 value in some cell lines (37). Here, in both T cell function testing in co-culture model and cervical cancer lines, our synthesized SP-PROTAC compound exerted a much more potent inhibition function than that of BMS-8, especially in cancer cells, and the IC50 value of BMS-8 was more than 144–170-fold the DC50 value of the SP-PROTAC compound. Therefore, our stapled peptide may play an important role in anti-PD-1/PD-L1 pathway. Although ICIs have shown clinically significant effects in some patients with cancer, the response rate for patients with cervical cancer remains very low. In some clinical studies on patients with advanced cervical cancer, the objective response rate was not satisfactory (50, 51). To date, no small molecule inhibitors have been approved for clinical use. Considering our findings regarding safety and efficacy, the SP-PROTAC compound may have significant potential for clinical development in treating cervical cancer compared to the existing PD-L1 inhibitor.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Institutional Ethics Board of Zhongnan Hospital of Wuhan University (Wuhan, Hubei, China, No. 2020029). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

M-YD initiated and conceived the project and designed the Peptide-PROTAC drugs. H-BC wrote the manuscript and critically provided strategical advice. M-YD and Y-YS designed and performed most of the experiments and interpreted data. A-JW and X-LL provided the technical and material support. All authors contributed to the article and approved the submitted version.