Ildikó Szabó

Background/Objectives: Longer oligoarginines are very effective cell-penetrating peptides. It has been shown that a minimal number of positively charged side chains is necessary for efficient cellular uptake. But a highly positively charged peptide may interact with its cargo molecule, thereby reducing its efficiency. Several chemical modifications were tested to improve the internalization of short tetraarginine derivatives. Aromatic groups, such as Dabcyl at the N-terminus, Trp in the sequence, and AMBA or PABA in the backbone, were used to improve internalization. The other useful modification was the aza-glycine substitution in the case of penetratin. Methods: In this study, the effect of aza-glycine insertion into the peptide Dabcyl-RRRRK(Cf) on internalization was studied and compared with that of the Trp-modified peptide Dabcyl-RRWRRK(Cf). To explain the noticed difference in the biological activity of peptides, DFT calculations and the prediction of membrane-binding free energy (ΔΔF) from a peptide sequence were performed. Results: It turned out that the position of the aza-glycine moiety does not have an influence on the cellular uptake. The aza-glycine-containing peptide showed higher internalization than the Dabcyl-RRRRK(Cf) peptide. Besides this, these peptides have similar or higher cellular uptake than that of octaarginine at lower concentrations (c < 2 µM). The aza-glycine affected not only cellular uptake but also the entry mechanism. The structure of peptides depended on the amino acids (Trp, Gly, or azaGly) in their sequences and their positions. Conclusions: These may result in the different amphiphilicity of peptides, and thus changes in the hydrophobic moment and in the binding affinity of peptides to the negatively charged membrane surface.

Background/Objectives: Investigating the modified derivatives of known cell-penetrating peptides can highlight the important residues in the peptide sequence and help understand the cellular uptake mechanism better. Moreover, comparing peptides with different fluorescent-dye positions can highlight the importance of the conjugation site. Earlier, it was demonstrated that the fluorescence quencher 4-((4-(dimethylamino)phenyl)azo)benzoyl (Dabcyl) group can enhance the internalization efficiency of highly cationic oligoarginine peptides. However, its effect in the case of arginine-rich penetratin, a secondary amphipathic cationic CPP, remains undiscovered. Methods: Here, several penetratin derivatives were studied in which the aromatic residues were substituted and the effect of Dabcyl modification was also studied on the cellular uptake of peptides by flow cytometry. Results: The triple Nal-substituted penetratin and dodeca-penetratin with N-terminally positioned carboxyfluoresein (Cf) dye demonstrated remarkable internalization efficiency compared to penetratin. Moreover, almost all the Dabcyl-modified peptides were superior to penetratin except two peptides with C-terminal Cf-labelling. This result highlights the importance of the structure of the conjugate. The position of the cargo molecule may have a high impact on internalization ability. The relatively low cellular uptake of the Trp48 residue-substituted Dabcyl-Pen12 points to the importance of this residue in the cellular uptake of dodeca-penetratin. The confocal microscopic studies revealed that, besides the greater penetration efficiency of Dabcyl penetratin derivatives, these peptides enter the cytoplasm of cells in an increased manner. Conclusions: We identified several intriguing derivatives and expanded the applicability of Dabcyl, while also highlighting its limitations. Additionally, the critical role of Trp48 in the penetratin sequence was reaffirmed, along with the importance of the fluorescent molecule’s position.

Getting through the cell membrane is challenging, and transporting a therapeutic agent while entering the cell is even more complicated. Cell-penetrating peptides (CPPs) are valuable tools for solving this problem, although they have drawbacks. In this work, the synthesis and investigation of efficient CPPs are described. We used an aromatic group, 1-pyrene carboxylic acid (PCA), to enhance internalization. We designed oligoarginines to investigate the effect of PCA in different positions at the N-terminus or in the side chain. Our novel peptide derivatives showed remarkable internalization on tumor cell lines, and more than one endocytic pathway plays a role in their internalization mechanism. With this modification, there is an opportunity to design short oligoarginines that can rival well-known CPPs like octaarginine in internalization.

Malignant melanoma is one of the most aggressive and resistant tumor types, with high metastatic properties. Because of the lack of suitable chemotherapeutic agents for treatment, the 5-year survival rate of melanoma patients with regional and distant metastases is lower than 10%. Targeted tumor therapy that provides several promising results might be a good option for the treatment of malignant melanomas. Our goal was to develop novel melanoma-specific peptide–drug conjugates for targeted tumor therapy. Melanocortin-1-receptor (MC1R) is a cell surface receptor responsible for melanogenesis and it is overexpressed on the surface of melanoma cells, providing a good target. Its native ligand, α-MSH (α-melanocyte-stimulating hormone) peptide, or its derivatives, might be potential homing devices for this purpose. Therefore, we prepared three α-MSH derivative–daunomycin (Dau) conjugates and their in vitro and in vivo antitumor activities were compared. Dau has an autofluorescence property; therefore, it is suitable for preparing conjugates for in vitro (e.g., cellular uptake) and in vivo experiments. Dau was attached to the peptides via a non-cleavable oxime linkage that was applied efficiently in our previous experiments, resulting in conjugates with high tumor growth inhibition activity. The results indicated that the most promising conjugate was the compound in which Dau was connected to the side chain of Lys (Ac-SYSNleEHFRWGK(Dau=Aoa)PV-NH₂). The highest cellular uptake by melanoma cells was demonstrated using the compound, with the highest tumor growth inhibition detected both on mouse (38.6% on B16) and human uveal melanoma (55% on OMC-1) cells. The effect of the compound was more pronounced than that of the free drug.

Malignant melanoma is one of the most aggressive and resistant tumor types which is often accompanied by poor prognosis when spread to nearby or distant tissues. Even though an increasing number of second-line therapies are available for cancer patients, the 5-year survival rate of melanoma patients with regional and distant metastases is still rather low. Therefore, there is an urgent need to develop new, highly efficient treatments. Melanocortin-1-receptor (MC1R) is a cell surface receptor for α-MSH. It is responsible for melanogenesis and is shown to be overexpressed on the surface of cancer cells in a subset of melanoma patients. Here, we designed and synthesized α-MSH analogs and conjugated them to daunomycin through oxime linkage. Using our peptide-drug conjugates (Dau-α-MSHs), we performed comparative in vitro and in vivo evaluations to reveal their suitability for tumor targeting. Firstly, the expression level of MC1R was determined in various melanoma cell lines via RT-qPCR. Then, we examined the antiproliferative effect of our Dau-α-MSHs, moreover we determined the cellular uptake profile and intracellular localization of Dau-α-MSHs. The most efficient Dau-α-MSHs were investigated in vivo using a murine xenograft melanoma model. We found that various cell lines show differential expression on the mRNA levels. We also showed that healthy primary cells have lower expression of MC1R compared to tumor cell lines. Moreover, the concentration-dependent cellular uptake profile of Dau-α-MSHs was detected and the compounds exhibited a great antiproliferative effect on melanoma cell lines. We also provided evidence that our novel Dau-α-MSHs can inhibit tumor expansion significantly and are suitable for targeted tumor therapy.

Delivering therapeutic agents into cells has always been a major challenge. In recent years, cyclization emerged as a tool for designing CPPs to increase their internalization and stability. Cyclic ring(s) can protect the peptide from enzymatic degradation, so cyclic peptides remain intact. Therefore they can be good carrier molecules. In this work, the preparation and investigation of efficient cyclic CPPs are described. Different oligoarginines were designed to conjugate with rigid aromatic scaffolds or form disulfide bonds. The reaction between the scaffolds and the peptides forms stable thioether bonds, constraining the peptide into a cyclic structure. The constructs presented very efficient internalization on cancerous cell lines. Our peptides use more than one endocytic pathway for cellular uptake. In this way, short peptides, which can compete with the penetration of well-known CPPs such as octaarginine (Arg₈), may be synthesized through cyclization.

Cell-penetrating peptides (CPPs) are commonly modified to increase their cellular uptake, alter the mechanism of penetration or enhance their endosomal release. Earlier, we described the internalization enhancement ability of the 4-((4-(dimethylamino)phenyl)azo)benzoyl (Dabcyl) group. We proved that this modification on the N-terminus of tetra- and hexaarginine enhanced their cellular uptake. The introduction of an aromatic ring 4-(aminomethyl) benzoic acid, AMBA) into the peptide backbone has a synergistic effect with Dabcyl, and the tetraarginine derivatives had outstanding cellular uptake. Based on these results, the effect of Dabcyl or Dabcyl-AMBA modification on the internalization of oligoarginines was studied. Oligoarginines were modified with these groups and their internalization was measured using flow cytometry. The concentration dependence of the cellular uptake of selected constructs was compared too. Their internalization mechanism was also examined by using different endocytosis inhibitors. While the effect of the Dabcyl group was optimal for hexaarginine, the Dabcyl-AMBA group increased the cellular uptake in the case of all oligoarginines. All derivatives, with the exception of only tetraarginine, were more effective than the octaarginine control. The internalization mechanism was dependent on the size of the oligoarginine and was independent of the modification. Our findings suggest that these modifications enhanced the internalization of oligoarginines and resulted in novel, very effective CPPs.

Cancer of the skin is by far the most common of all cancers. Although the incidence of melanoma is relatively low among skin cancers, it can account for a high number of skin cancer deaths. Since the start of deeper insight into the mechanisms of melanoma tumorigenesis and their strong interaction with the immune system, the development of new therapeutical strategies has been continuously rising. The high number of melanoma cell mutations provides a diverse set of antigens that the immune system can recognize and use to distinguish tumor cells from normal cells. Peptide-based synthetic anti-tumor vaccines are based on tumor antigens that elicit an immune response due to antigen-presenting cells (APCs). Although targeting APCs with peptide antigens is the most important assumption for vaccine development, peptide antigens alone are poorly immunogenic. The immunogenicity of peptide antigens can be improved not only by synthetic modifications but also by the assistance of adjuvants and/or delivery systems. The current review summarizes the different chemical approaches for the development of effective peptide-based vaccines for the immunotherapeutic treatment of advanced melanoma.

Utilizing McMurry reactions of 4,4′-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic activity was evaluated in vitro on four human malignant cell lines (MCF-7, MDA-MB 231, A2058, HT-29). It was found that some of these novel Tamoxifen analogues show marked cytotoxicity in a dose-dependent manner. The relative ROS-generating capability of the synthetized analogues was evaluated by cyclic voltammetry (CV) and DFT modeling studies. The results of cell-viability assays, CV measurements and DFT calculations suggest that the cytotoxicity of the majority of the novel compounds is mainly elicited by their interactions with cellular targets including estrogen receptors rather than triggered by redox processes. However, three novel compounds could be involved in ROS-production and subsequent formation of quinone-methide preventing proliferation and disrupting the redox balance of the treated cells. Among the cell lines studied, HT-29 proved to be the most susceptible to the treatment with compounds having ROS-generating potency.

Cell-penetrating peptides (CPP) are promising tools for the transport of a broad range of compounds into cells. Since the discovery of the first members of this peptide family, many other peptides have been identified; nowadays, dozens of these peptides are known. These peptides sometimes have very different chemical–physical properties, but they have similar drawbacks; e.g., non-specific internalization, fast elimination from the body, intracellular/vesicular entrapment. Although our knowledge regarding the mechanism and structure–activity relationship of internalization is growing, the prediction and design of the cell-penetrating properties are challenging. In this review, we focus on the different modifications of well-known CPPs to avoid their drawbacks, as well as how these modifications may increase their internalization and/or change the mechanism of penetration.