Design, Synthesis, and Antitumor Evaluation of an Opioid Growth Factor Bioconjugate Targeting Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) presents a formidable challenge with high lethality and limited effective drug treatments. Its heightened metastatic potential further complicates the prognosis. Owing to the significant toxicity of current chemotherapeutics, compounds like [Met5]-enkephalin, known as opioid growth factor (OGF), have emerged in oncology clinical trials. OGF, an endogenous peptide interacting with the OGF receptor (OGFr), plays a crucial role in inhibiting cell proliferation across various cancer types. This in vitro study explores the potential anticancer efficacy of a newly synthesized OGF bioconjugate in synergy with the classic chemotherapeutic agent, gemcitabine (OGF-Gem). The study delves into assessing the impact of the OGF-Gem conjugate on cell proliferation inhibition, cell cycle regulation, the induction of cellular senescence, and apoptosis. Furthermore, the antimetastatic potential of the OGF-Gem conjugate was demonstrated through evaluations using blood platelets and AsPC-1 cells with a light aggregometer. In summary, this article demonstrates the cytotoxic impact of the innovative OGF-Gem conjugate on pancreatic cancer cells in both 2D and 3D models. We highlight the potential of both the OGF-Gem conjugate and OGF alone in effectively inhibiting the ex vivo pancreatic tumor cell-induced platelet aggregation (TCIPA) process, a phenomenon not observed with Gem alone. Furthermore, the confirmed hemocompatibility of OGF-Gem with platelets reinforces its promising potential. We anticipate that this conjugation strategy will open avenues for the development of potent anticancer agents.

Lactoferricin B Combined with Antibiotics Exhibits Leukemic Selectivity and Antimicrobial Activity.

The fusion of penetrating peptides (PPs), e.g., cell penetration peptides (CPPs) or antimicrobial peptides (AMPs), together with antimicrobial agents is an expanding research field. Specific AMPs, such as lactoferricin B (LfcinB), have demonstrated strong antibacterial, antifungal, and antiparasitic activity, as well as valuable anticancer activity, proving beneficial in the development of anticancer conjugates. The resulting conjugates offer potential dual functionality, acting as both an anticancer and an antimicrobial agent. This is especially necessary in cancer treatment, where microbial infections pose a critical risk. Leukemic cells frequently exhibit altered outer lipid membranes compared to healthy cells, making them more sensitive to compounds that interfere with their membrane. In this study, we revisited and reanalyzed our earlier research on LfcinB and its conjugates. Furthermore, we carried out new experiments with a specific focus on cell proliferation, changes in membrane asymmetric phosphatidylserine location, intracellular reactive oxygen species (ROS) generation, mitochondrial functions, and in vitro bacterial topoisomerase inhibition.

Synthesis of Novel Arginine Building Blocks with Increased Lipophilicity Compatible with Solid-Phase Peptide Synthesis.

Arginine, due to the guanidine moiety, increases peptides' hydrophilicity and enables interactions with charged molecules, but at the same time, its presence in a peptide chain might reduce its permeability through biological membranes. This might be resolved by temporary coverage of the peptide charge by lipophilic, enzyme-sensitive alkoxycarbonyl groups. Unfortunately, such a modification of a guanidine moiety has not been reported to date and turned out to be challenging. Here, we present a new, optimized strategy to obtain arginine building blocks with increased lipophilicity that were successfully utilized in the solid-phase peptide synthesis of novel arginine vasopressin prodrugs.

Dual-Activity Fluoroquinolone-Transportan 10 Conjugates Offer Alternative Leukemia Therapy during Hematopoietic Cell Transplantation.

Hematopoietic cell transplantation (HCT) is often considered a last resort leukemia treatment, fraught with limited success due to microbial infections, a leading cause of mortality in leukemia patients. To address this critical issue, we explored a novel approach by synthesizing antileukemic agents containing antibacterial substances. This innovative strategy involves conjugating fluoroquinolone antibiotics, such as ciprofloxacin (CIP) or levofloxacin (LVX), with the cell-penetrating peptide transportan 10 (TP10). Here, we demonstrate that the resultant compounds display promising biologic activities in preclinical studies. These novel conjugates not only exhibit potent antimicrobial effects but are also selective against leukemia cells. The cytotoxic mechanism involves rapid disruption of cell membrane asymmetry leading to membrane damage. Importantly, these conjugates penetrated mammalian cells, accumulating within the nuclear membrane without significant effect on cellular architecture or mitochondrial function. Molecular simulations elucidated the aggregation tendencies of TP10 conjugates within lipid bilayers, resulting in membrane disruption and permeabilization. Moreover, mass spectrometry analysis confirmed efficient reduction of disulfide bonds within TP10 conjugates, facilitating release and activation of the fluoroquinolone derivatives. Intriguingly, these compounds inhibited human topoisomerases, setting them apart from traditional fluoroquinolones. Remarkably, TP10 conjugates generated lower intracellular levels of reactive oxygen species compared with CIP and LVX. The combination of antibacterial and antileukemic properties, coupled with selective cytostatic effects and minimal toxicity toward healthy cells, positions TP10 derivatives as promising candidates for innovative therapeutic approaches in the context of antileukemic HCT. This study highlights their potential in search of more effective leukemia treatments. SIGNIFICANCE STATEMENT: Fluoroquinolones are commonly used antibiotics, while transportan 10 (TP10) is a cell-penetrating peptide (CPP) with anticancer properties. In HCT, microbial infections are the primary cause of illness and death. Combining TP10 with fluoroquinolones enhanced their effects on different cell types. The dual pharmacological action of these conjugates offers a promising proof-of-concept solution for leukemic patients undergoing HCT. Strategically designed therapeutics, incorporating CPPs with antibacterial properties, have the potential to reduce microbial infections in the treatment of malignancies.

Cyclic Peptidic Furin Inhibitors Developed by Combinatorial Chemistry.

Furin is a human serine protease responsible for activating numerous physiologically relevant cell substrates and is also involved in the development of various pathological conditions, including inflammatory diseases, cancers, and viral and bacterial infections. Therefore, compounds with the ability to inhibit furin's proteolytic action are regarded as potential therapeutics. Here we took the combinatorial chemistry approach (library consisting of 2000 peptides) to obtain new, strong, and stable peptide furin inhibitors. The extensively studied trypsin inhibitor SFTI-1 was used as a leading structure. A selected monocylic inhibitor was further modified to finally yield five mono- or bicyclic furin inhibitors with values of K i in the subnanomolar range. Inhibitor 5 was the most active (K i = 0.21 nM) and significantly more proteolytically resistant than the reference furin inhibitor described in the literature. Moreover, it reduced furin-like activity in PANC-1 cell lysate. Detailed analysis of furin-inhibitor complexes using molecular dynamics simulations is also reported.

Effective Drug Concentration and Selectivity Depends on Fraction of Primitive Cells.

Poor efficiency of chemotherapeutics in the eradication of Cancer Stem Cells (CSCs) has been driving the search for more active and specific compounds. In this work, we show how cell density-dependent stage culture profiles can be used in drug development workflows to achieve more robust drug activity (IC50 and EC50) results. Using flow cytometry and light microscopy, we characterized the cytological stage profiles of the HL-60-, A-549-, and HEK-293-derived sublines with a focus on their primitive cell content. We then used a range of cytotoxic substances-C-123, bortezomib, idarubicin, C-1305, doxorubicin, DMSO, and ethanol-to highlight typical density-related issues accompanying drug activity determination. We also showed that drug EC50 and selectivity indices normalized to primitive cell content are more accurate activity measurements. We tested our approach by calculating the corrected selectivity index of a novel chemotherapeutic candidate, C-123. Overall, our study highlights the usefulness of accounting for primitive cell fractions in the assessment of drug efficiency.

Cu(II) complexes with peptides from FomA protein containing -His-Xaa-Yaa-Zaa-His and -His-His-motifs. ROS generation and DNA degradation.

Mono- and dinuclear Cu(II) complexes with Ac-PTVHNEYH-NH2 (L1) and Ac-NHHTLND-NH2 (L2) peptides from FomA protein of Fusobacterium nucleatum were studied by potentiometry, spectroscopic methods (UV-Vis, CD, EPR) and MS technique. The dominant mononuclear complexes for L1 ligand are: CuHL (pH range 5.0-6.0) with 2N {2Nim}, CuH-2L (pH range 8.0-8.5) and CuH-3L species (above pH 9.0) with 4N {Nim, 3N-} coordination modes. The complexes: CuH-1L with 3N {2Nim, N-}, CuH-2L with 3N {Nim, 2N-} and CuH-3L with 4N {Nim, 3N-} binding sites are proposed for the L2 ligand. Probably in the CuH-2L complex for CuL2 system the second His residue in His-His sequence is bound to Cu(II) ion, while the first His residue may stabilize this complex by His-His and/or His-Cu(II) interactions. The dominant dinuclear Cu2L1 complexes in the pH range 6.5-10.5 are: the Cu2H-4L and Cu2H-6L species with 3N{Nim, 2N-}4N{Nim, 3N-} and 4N{Nim, 3N-}4N{Nim, 3N-} binding sites, respectively. In the case of the Cu2L2 complex in the pH range 7.2-10.5, the Cu2H-4L and Cu2H-7L species dominate with 2N{Nim, N-}4N{Nim, 3N-} and (Cu(OH)42-4N{Nim, 3N-}) coordination modes, respectively. The ability to generate reactive oxygen species (ROS) by uncomplexed Cu(II) ions, ligands and their complexes at pH 7.4 in the presence of hydrogen peroxide or ascorbic acid was studied. UV-Vis, luminescence, EPR spin trapping and gel electrophoresis methods were used. Both complexes produce higher level of ROS compared to those of their ligands. ROS produced by Cu(II) complexes are hydroxyl radical and singlet oxygen, which contribute to oxidative DNA cleavage.

Conjugates of Ciprofloxacin and Levofloxacin with Cell-Penetrating Peptide Exhibit Antifungal Activity and Mammalian Cytotoxicity.

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH2) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH2 resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the Candida genus (MICs in the 6.25 - 100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH2 was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH2 and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC50 values in the 10 - 100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH2 since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable.

Mushroom extracts and compounds with suppressive action on breast cancer: evidence from studies using cultured cancer cells, tumor-bearing animals, and clinical trials.

This article reviews mushrooms with anti-breast cancer activity. The mushrooms covered which are better known include the following: button mushroom Agaricus bisporus, Brazilian mushroom Agaricus blazei, Amauroderma rugosum, stout camphor fungus Antrodia camphorata, Jew's ear (black) fungus or black wood ear fungus Auricularia auricula-judae, reishi mushroom or Lingzhi Ganoderma lucidum, Ganoderma sinense, maitake mushroom or sheep's head mushroom Grifola frondosa, lion's mane mushroom or monkey head mushroom Hericium erinaceum, brown beech mushroom Hypsizigus marmoreus, sulfur polypore mushroom Laetiporus sulphureus, Lentinula edodes (shiitake mushroom), Phellinus linteus (Japanese "meshimakobu," Chinese "song gen," Korean "sanghwang," American "black hoof mushroom"), abalone mushroom Pleurotus abalonus, king oyster mushroom Pleurotus eryngii, oyster mushroom Pleurotus ostreatus, tuckahoe or Fu Ling Poria cocos, and split gill mushroom Schizophyllum commune. Antineoplastic effectiveness in human clinical trials and mechanism of anticancer action have been reported for Antrodia camphorata, Cordyceps sinensis, Coriolus versicolor, Ganoderma lucidum, Grifola frondosa, and Lentinula edodes.

Truncation of Huia versabilis Bowman-Birk inhibitor increases its selectivity, matriptase-1 inhibitory activity and proteolytic stability.

A gradual truncation of the primary structure of frog skin-derived Huia versabilis Bowman-Birk peptidic inhibitor (HV-BBI) resulted in 18-times stronger inhibitor of matriptase-1 (peptide 6, Ki = 8 nm) in comparison to the full-length HV-BBI (Ki = 155 nm). Analogous increase in the inhibitory activity in correlation with the peptide length reduction was not observed in case of other serine proteases, bovine trypsin (Ki = 151 nm for peptide 6 and Ki = 120 nm for HV-BBI) and plasmin (Ki = 120 nm for peptide 6 and 82 nm for HV-BBI). Weaker binding affinity to these enzymes emphasized an inhibitory specificity of peptide 6. Molecular dynamic analysis revealed that the observed variations in the binding affinity of peptide 6 and HV-BBI with matriptase-1 are associated with the entropic differences of the unbound peptides. Moreover, several aspects explaining differences in the inhibition of matriptase-1 by peptide 6 (bearing the C-terminal amide group) and its two analogues, peptide 6∗ (having the C-terminal carboxyl group, Ki = 473 nm) and cyclic peptide 6∗∗ (Ki = 533 nm), both exhibiting more than 50-fold reduced inhibitory potency, were discovered. It was also shown that peptide 6 presented significantly higher resistance to proteolytic degradation in human serum than HV-BBI. Additional investigations revealed that, in contrast to some amphibian-derived inhibitors, HV-BBI and its truncated analogues do not possess bactericidal activity, thus they cannot be considered as bifunctional agents.

New ribosome-inactivating proteins and other proteins with protein synthesis-inhibiting activities.

Ribosome-inactivating proteins (RIPs) consist of three varieties. Type 1 RIPs are single-chained and approximately 30-kDa in molecular weight. Type 2 RIPs are double-chained and composed of a type 1 RIP chain and a lectin chain. Type III RIPs, such as maize b-32 barley and JIP60 which are produced as single-domain proenzymes, possess an N-terminal domain corresponding to the A domain of RIPs and fused to a C-terminal domain. In addition to the aforementioned three types of RIPs originating from flowering plants, there are recently discovered proteins and peptides with ribosome-inactivating and protein synthesis inhibitory activities but which are endowed with characteristics such as molecular weights distinctive from those of the regular RIPs. These new/unusual RIPs discussed in the present review encompass metazoan RIPs from Anopheles and Culex mosquitos, antimicrobial peptides derived from RIP of the pokeweed Phytolacca dioica, maize RIP (a type III RIP derived from a precursor form), RIPs from the garden pea and the kelp. In addition, RIPs with a molecular weight smaller than those of regular type 1 RIPs are produced by plants in the Cucurbitaceae family including the bitter gourd, bottle gourd, sponge gourd, ridge gourd, wax gourd, hairy gourd, pumpkin, and Chinese cucumber. A small type II RIP from camphor tree (Cinnamomum camphora) seeds and a snake gourd type II RIP with its catalytic chain cleaved into two have been reported. RIPs produced from mushrooms including the golden needle mushroom, king tuber mushroom, straw mushroom, and puffball mushroom are also discussed in addition to a type II RIP from the mushroom Polyporus umbellatus. Bacterial (Spiroplasma) RIPs associated with the fruitfly, Shiga toxin, and Streptomyces coelicolor RIP are also dealt with. The aforementioned proteins display a diversity of molecular weights, amino acid sequences, and mechanisms of action. Some of them are endowed with exploitable antipathogenic activities.

Antibiotic-Based Conjugates Containing Antimicrobial HLopt2 Peptide: Design, Synthesis, Antimicrobial and Cytotoxic Activities.

Recent studies have shown that modified human lactoferrin 20-31 fragment, named HLopt2, possesses antibacterial and antifungal activity. Thus, we decided to synthesize and evaluate the biological activity of a series of conjugates based on this peptide and one of the antimicrobials with proven antibacterial (ciprofloxacin, CIP, and levofloxacin, LVX) or antifungal (fluconazole, FLC) activity. The drugs were covalently connected to the peptide via amide, methylenecarbonyl moieties, or a disulfide bridge. The antibacterial and antifungal activities were evaluated under Clinical and Laboratory Standard Institute (CLSI) recommended conditions or in a low-salt brain-heart infusion diluted medium (BHI1/100). Results showed that conjugation of the peptide with the drug increased its antimicrobial activity up to 4-fold. Under CLSI-recommended conditions, all the compounds revealed rather low efficiency. Among conjugates, the highest antibacterial activity was recorded for the CIP-Cys-S-S-HLopt2-NH2 (III). In BHI1/100, which had lower differentiating properties, all of the conjugates revealed low MIC and MMC (minimum inhibitory and microbicidal concentrations) values. The disulfide bridge used as a linker in the most active conjugate (III) upon incubation with S. aureus cells is reduced, releasing constituent peptide and CIP-Cys. In addition, we showed that its fluorescently labeled analogue and constituent peptide are able to be internalized into both C. albicans and S. aureus cells. Moreover, the invaluable advantage of the presented conjugates was their low toxicity to mammalian cells and very low hemolytic activity. The current research can form a solid basis for further in vivo studies and drug development.

Peptide conjugates of lactoferricin analogues and antimicrobials-Design, chemical synthesis, and evaluation of antimicrobial activity and mammalian cytotoxicity.

Eight new peptide conjugates composed of modified bovine lactoferricin truncated analogues (LFcinB) and one of the three antimicrobials - ciprofloxacin (CIP), levofloxacin (LVX), and fluconazole (FLC) - were synthesized. Four different linkers were applied to connect a peptide and an antimicrobial agent. The FLC-containing peptidic conjugates were synthesized using the "click chemistry" method. This novel approach is reported here for the first time. Unlike their components, CIP- and LVX-based conjugates exerted activity against Candida yeast. Similarly to the constituent peptides, synthesized conjugates showed activity against Gram-positive bacteria, especially S. epidermidis. The most active were the conjugates containing CIP linked to the peptide by the redox-sensitive disulfide bridge. Our results show a significant role of a linker between antimicrobial agent and a peptide. This was also confirmed by the lack of synergistic effects on the antimicrobial activity of the constituent compounds. Moreover, cytotoxicity assays revealed that the proposed conjugates cause a comparatively low cytotoxic effect in reference to antibiotics widely used in therapies. Therefore, they can be deliberated as attractive leading structures for the development of drugs.

Matriptase-2: monitoring and inhibiting its proteolytic activity.

Matriptase-2 (MT2) is a membrane-anchored proteolytic enzyme. It acts as the proteolytic key regulator in human iron homeostasis. A high expression level can lead to iron overload diseases, whereas mutations in the gene encoding MT2, TMPRSS6, may result in various forms of iron deficiency anemia. Recently, MT2 has been reported as a positive prognostic factor in breast and prostate cancers. However, the exact functions of MT2 in various pathophysiological conditions are still not fully understood. In this review, we describe the synthetic tools designed and synthesized to regulate or monitor MT2 proteolytic activity and present the latest knowledge about the role of MT2 in iron homeostasis and cancer.

Copper(II) complexes with Fusobacterium nucleatum adhesin FadA: Coordination pattern, physicochemical properties and reactivity.

Fusobacterium nucleatum is an anaerobic, Gram-negative bacteria linked to colon cancer. It is interesting to determine how metal ions interact with bacterial adhesin proteins. To this end, the coordination of ATDAAS-NH2 and MKKFL-NH2 fragments of Fusobacterium adhesin A (FadA) to copper(II) ions was studied by potentiometry, spectroscopic techniques (UV-Vis, CD, EPR and NMR) and the density functional theory (DFT) methods. At pH 6.8 (colon physiological pH), the metal ion in the first peptide (ATDAAS-NH2) is coordinated by one oxygen and three nitrogen donors while in the second one (MKKFL-NH2) - by sulfur and three nitrogen atoms. Both complexes form two five- and one six-membered stable chelate rings. Moreover, reactivity studies confirmed the production of reactive oxygen species such as hydroxyl radical, superoxide anion radical and singlet oxygen. Generation of reactive oxygen species (ROS) was observed during gel electrophoresis and spectroscopic assays with reporting molecules like NDMA (N,N-dimethyl-p-nitrosoaniline) and NBT (Nitrotetrazolium Blue Chloride). All reactions were conducted in the presence of hydrogen peroxide as endogenous oxidant.

Antimicrobial Activity of Chimera Peptides Composed of Human Neutrophil Peptide 1 (HNP-1) Truncated Analogues and Bovine Lactoferrampin.

Three chimera peptides composed of bovine lactoferrampin and the analogue of truncated human neutrophil peptide 1 were synthesized by the solid-phase method. In two compounds peptide chains were connected via isopeptide bond, whereas in the third one disulfide bridge served as a linker. All three chimeras displayed significantly higher antimicrobial activity than the constituent peptides as well as their equimolar mixtures. The one with a disulfide bridge displayed selectivity toward Gram-positive bacteria and was able to penetrate bacterial cells. The chimeric peptides demonstrated low in vitro mammalian cytotoxicity, especially against benign cells. The significance of linker type was also reflected in the secondary structure and proteolytic stability of studied compounds. Presented results proved that such chimeras are good lead structures for designing antimicrobial drugs.

Tuber Lectins with Potentially Exploitable Bioactivities.

Lectins are a group of proteins or glycoproteins with various potentially exploitable bioactivities and have been capturing more interest recently. They have been isolated and reported from various tissues of a diversity of plant species. Tubers are modified and enlarged plant structures derived from stems or roots that are used for nutrient storage and asexual reproduction. A number of plants such as yam, taro and potato are grown for their edible tubers, and lectins are found to be one of the major storage proteins. These lectins exhibit potent bioactivities encompassing mitogenic, antitumor, antimicrobial, immunomodulatory, antioxidative, hypoglycemic, insecticidal and nematicidal activities. They are potential resources for development into functional or healthy foods and targets for food protein researchers.

Spliced analogues of trypsin inhibitor SFTI-1 and their application for tracing proteolysis and delivery of cargos inside the cells.

A series of analogues of trypsin inhibitor SFTI-1 were designed and synthesized to monitor peptide splicing. In the middle part of the SFTI-1 analogues, which is released upon incubation with proteinase, the RGD sequence or an acceptor of fluorescence for FRET was introduced. The results of studies with trypsin confirmed that the designed analogues underwent peptide splicing. Furthermore, we showed that a FRET displaying SFTI-1 analogue was internalized into the HaCaT keratinocytes, where it was degraded. Therefore, both proteolysis and the reduction of the disulfide bridge of the peptide took place. As a result, such analogues are a convenient tool to trace the proteolytic activity inside the cell. However, the cytotoxicity of SFTI-1 analogues grafted with the RGD sequence did not correlate with their susceptibility to peptide splicing. Nevertheless, these peptides were slightly more active than the reference peptide (GRGDNP). Interestingly, one of the analogues assigned as [desSer6 ]VI, under experimental conditions, appeared significantly more cytotoxic towards cancer cells U87-MG in contrast to the reference peptide.

Activities of Venom Proteins and Peptides with Possible Therapeutic Applications from Bees and WASPS.

The variety of proteins and peptides isolated from honey bee venom and wasp venom includes melittin, adiapin, apamine, bradykinin, cardiopep, mast cell degranulating peptide, mastoparan, phospholipase A2 and secapin. Some of the activities they demonstrate may find therapeutic applications.

Noncovalent inhibitors of human 20S and 26S proteasome based on trypsin inhibitor SFTI-1.

Sunflower trypsin inhibitor (SFTI-1) is recognized as an attractive scaffold to designed potent inhibitors of various proteases. We have recently found that its analogues inhibit noncovalently both human and yeast 20S proteasomes. Here, a set of novel and more potent in vitro inhibitors is presented. The inhibitory potency of the peptides was assessed with human 20S proteasome in the presence or absence of sodium dodecyl sulfate and with human 26 proteasome. Their antiproliferative action against tumor (human melanoma cells A375) and normal cells (46 BR.1N human fibroblasts and HaCaT keratinocytes) was determined. The selected fluoresceine-labeled inhibitors were able to internalize into A375 cells and were sometimes present as foci in the cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 685-696, 2016.