Redox-Responsive Disulfide Cyclic Peptides: A New Strategy for siRNA Delivery.

RNA interference (RNAi) is a powerful tool capable of targeting virtually any protein without time-consuming and expensive drug development studies. However, due to obstacles facing efficient and safe delivery, RNAi-based therapeutic approach remains a challenge. Herein, we have designed and synthesized a number of disulfide-constraining cyclic and hybrid peptides using tryptophan and arginine residues. Our hypothesis was that peptide structures would undergo reduction by intracellular glutathione (more abundant in cancer cells) and unpack the small interfering RNA (siRNA) from the peptide/siRNA complexes. A subset of newly developed peptides (specifically, C4 and H4) exhibited effective cellular internalization of siRNA (∼70% of the cell population; monitored by flow cytometry and confocal microscopy), the capability of protecting siRNA against early degradation by nucleases (monitored by gel electrophoresis), minimal cytotoxicity in selected cell lines (studied by cell viability and LC50 calculations), and efficient protein silencing by 70-75% reduction in the expression of targeting signal transducer and activator of transcription 3 (STAT3) in human triple-negative breast cancer (TNBC) MDA-MB-231 cells, analyzed using the Western blot technique. Our results indicate the birth of a promising new family of siRNA delivery systems that are capable of safe and efficient delivery, even in the presence of nucleases.

siRNA Therapeutics for the Therapy of COVID-19 and Other Coronaviruses.

The ongoing pandemic of global concern has killed about three million humans and affected around 151 million people worldwide, as of April 30, 2021. Although recently approved vaccines for COVID-19 are engendering hope, finding new ways to cure the viral pandemic is still a quest for researchers worldwide. Major pandemics in history have been of viral origin, such as SARS, MERS, H1NI, Spanish flu, and so on. A larger emphasis has been on discovering potential vaccines, novel antiviral drugs, and agents that can mitigate the viral infection symptoms; however, a relatively new area, RNA interference (RNAi), has proven effective as an antiviral agent. The RNAi phenomenon has been largely exploited to cure cancer, neurodegenerative diseases, and some rare diseases. The U.S. Food and Drug Administration has recently approved three siRNA products for human use that garner significant hope in siRNA therapeutics for coronaviruses. There have been some commentaries and communications addressing this area. We have summarized and illustrated the significance and the potential of the siRNA therapeutics available as of April 30, 2021 to combat the ongoing viral pandemic and the emerging new variants such as B.1.1.7 and B.1.351. Numerous successful in vitro studies and several investigations to address the clinical application of siRNA therapeutics provide great hope in this field. This seminal Review describes the significance of siRNA-based therapy to treat diverse viral infections in addition to the current coronavirus challenge. In addition, we have thoroughly reviewed the patents approved for coronaviruses, the major challenges in siRNA therapy, and the potential approaches to address them, followed by innovation and prospects.

Cyclic Peptides as Protein Kinase Inhibitors: Structure-Activity Relationship and Molecular Modeling.

Under-expression or overexpression of protein kinases has been shown to be associated with unregulated cell signal transduction in cancer cells. Therefore, there is major interest in designing protein kinase inhibitors as anticancer agents. We have previously reported [WR]5, a peptide containing alternative arginine (R) and tryptophan (W) residues as a non-competitive c-Src tyrosine kinase inhibitor. A number of larger cyclic peptides containing alternative hydrophobic and positively charged residues [WR]x (x = 6-9) and hybrid cyclic-linear peptides, [R6K]W6 and [R5K]W7, containing R and W residues were evaluated for their protein kinase inhibitory potency. Among all the peptides, cyclic peptide [WR]9 was found to be the most potent tyrosine kinase inhibitor. [WR]9 showed higher inhibitory activity (IC50 = 0.21 µM) than [WR]5, [WR]6, [WR]7, and [WR]8 with IC50 values of 0.81, 0.57, 0.35, and 0.33 µM, respectively, against c-Src kinase as determined by a radioactive assay using [γ-33P]ATP. Consistent with the result above, [WR]9 inhibited other protein kinases such as Abl kinase activity with an IC50 value of 0.35 µM, showing 2.2-fold higher inhibition than [WR]5 (IC50 = 0.79 µM). [WR]9 also inhibited PKCa kinase activity with an IC50 value of 2.86 µM, approximately threefold higher inhibition than [WR]5 (IC50 = 8.52 µM). A similar pattern was observed against Braf, c-Src, Cdk2/cyclin A1, and Lck. [WR]9 exhibited IC50 values of <0.25 µM against Akt1, Alk, and Btk. These data suggest that [WR]9 is consistently more potent than other cyclic peptides with a smaller ring size and hybrid cyclic-linear peptides [R6K]W6 and [R5K]W7 against selected protein kinases. Thus, the presence of R and W residues in the ring, ring size, and the number of amino acids in the structure of the cyclic peptide were found to be critical in protein kinase inhibitory potency. We identified three putative binding pockets through automated blind docking of cyclic peptides [WR](5-9). The most populated pocket is located between the SH2, SH3, and N-lobe domains on the opposite side of the ATP binding site. The second putative pocket is formed by the same domains and located on the ATP binding site side of the protein. Finally, a third pocket was identified between the SH2 and SH3 domains. These results are consistent with the non-competitive nature of the inhibition displayed by these molecules. Molecular dynamics simulations of the protein-peptide complexes indicate that the presence of either [WR]5 or [WR]9 affects the plasticity of the protein and in particular the volume of the ATP binding site pocket in different ways. These results suggest that the second pocket is most likely the site where these peptides bind and offer a plausible rationale for the increased affinity of [WR]9.

Comparative Antiviral Activity of Remdesivir and Anti-HIV Nucleoside Analogs Against Human Coronavirus 229E (HCoV-229E).

Remdesivir is a nucleotide prodrug that is currently undergoing extensive clinical trials for the treatment of COVID-19. The prodrug is metabolized to its active triphosphate form and interferes with the action of RNA-dependent RNA polymerase of SARS-COV-2. Herein, we report the antiviral activity of remdesivir against human coronavirus 229E (HCoV-229E) compared to known anti-HIV agents. These agents included tenofovir (TFV), 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), alovudine (FLT), lamivudine (3TC), and emtricitabine (FTC), known as nucleoside reverse-transcriptase inhibitors (NRTIs), and a number of 5'-O-fatty acylated anti-HIV nucleoside conjugates. The anti-HIV nucleosides interfere with HIV RNA-dependent DNA polymerase and/or act as chain terminators. Normal human fibroblast lung cells (MRC-5) were used to determine the cytotoxicity of the compounds. The study revealed that remdesivir exhibited an EC50 value of 0.07 µM against HCoV-229E with TC50 of > 2.00 µM against MRC-5 cells. Parent NRTIs were found to be inactive against (HCoV-229E) at tested concentrations. Among all the NRTIs and 5'-O-fatty acyl conjugates of NRTIs, 5'-O-tetradecanoyl ester conjugate of FTC showed modest activity with EC50 and TC50 values of 72.8 µM and 87.5 µM, respectively. These data can be used for the design of potential compounds against other coronaviruses.

Design, Synthesis, and Evaluation of the Kinase Inhibition Potential of Pyridylpyrimidinylaminophenyl Derivatives.

In view of potent kinase inhibitors for the treatment of myriad human disorders, we synthesized some structurally variant amide/cyclic amide derivatives based on pyridylpyrimidinylaminophenyl amine, the key pharmacophore of the kinase inhibitor drug molecule, imatinib, and evaluated their kinase inhibition potency. Among the various synthesized amides, compound 20, a cyclic amide/pyridin-2(1H)-one derivative, exhibited an IC50 value comparable to that of the drug imatinib against c-Src kinase, and another compound (14) containing a 2-((4-methyl-2-oxo-2H-chromen-6-yl)oxy)acetamide demonstrated an IC50 value of 8.39 µM. Furthermore, the constitution of the cyclic amide derivative was confirmed by the single-crystal X-ray diffraction technique. These results may serve as a gateway for developing novel next-generation kinase inhibitors.

Regio- and Stereoselective Domino Synthesis of Oxazolo Fused Pyridoindoles and Benzofurooxazolo Pyridines from ortho-Alkynylarylaldehydes.

An environmentally benign Au(III)-catalyzed regio- and stereoselective domino synthesis of oxazolo fused pyridoindoles 7a-v and benzofurooxazolo pyridines 8a-n by the reaction of o-alkynylaldehydes 4a-t and 5a-k with (S)-phenylglycinol 6a and (R)-phenylglycinol 6b under mild reaction conditions using water as reaction medium is reported. The reaction proceeded via selective C-N bond formation on the more electrophilic alkynyl carbon through 6-endo-dig cyclization. The reaction tolerates a wide variety of functional groups. The developed chemistry has been successfully extended for the synthesis of a diverse class of γ-carbolines and benzofuro[3,2-c]pyridines using corresponding ester hydrochlorides of serine, threonine, and cystine as a nitrogen source.

Cysteine and arginine-rich peptides as molecular carriers.

A number of linear and cyclic peptides containing alternative arginine and cysteine residues, namely linear (CR)3, linear (CR)4, linear (CR)5, cyclic [CR]4, and cyclic [CR]5, were synthesized. The peptides were evaluated for their ability to deliver two molecular cargos, fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F'-GpYEEI) and fluorescence-labeled lamivudine (F'-3TC), intracellularly in human leukemia cancer (CCRF-CEM) cells. We investigated the role of cyclization and the number of amino acids in improving the transporting ability of the peptides. The flow cytometry studies suggested that the synthesized peptides were able to work efficiently as transporters for both cargos. Among all compounds, cyclic [CR]4 was found to be the most efficient peptide in transporting the cargo into cells. For instance, the cellular uptake of F'-3TC (5µM) and F'-GpYEEI (5µM) was enhanced by 16- and 20-fold, respectively, in the presence of cyclic [CR]4 compared to that of the parent compound alone. The mechanism of F'-GpYEEI uptake by cells was found to be energy-independent. The results showed that the number of amino acids and their cyclic nature can impact the efficiency of the peptide in transporting the molecular cargos.

Pd-catalyzed one-pot sequential unsymmetrical cross-coupling reactions of aryl/heteroaryl 1,2-dihalides.

Efficient, step-economic, Pd(ii)-catalyzed one-pot sequential Sonogashira/Sonogashira, Sonogashira/Suzuki, Sonogashira/Heck, Suzuki/Sonogashira, Suzuki/Suzuki, Suzuki/Heck, Heck/Sonogashira, Heck/Suzuki and Heck/Heck cross coupling reactions of sterically hindered aryl/heteroaryl 1,2-dihalides have been developed. The present methodology allows the conversion of easily available aryl/heteroaryl 1,2-dihalides into synthetically useful unsymmetrically substituted arenes/heteroarenes in good to excellent yields under mild reaction conditions. This methodology is a powerful tool for building a versatile substrate which can be utilized for the synthesis of various organic scaffolds.

Tandem Approach to Benzothieno- and Benzofuropyridines from o-Alkynyl Aldehydes via Silver-Catalyzed 6-endo-dig Ring Closure.

An operationally simple silver-catalyzed tandem strategy for the synthesis of benzothienopyridines 7a-t and benzofuropyridines 8a-p by the reaction of o-alkynyl aldehyde 4a-t and 5a-p with tert-butylamine 6 under mild reaction conditions is described. The present methodology provides a facile conversion of easily accessible o-alkynyl aldehydes into medicinally useful heterocycles in good to excellent yields under mild and environmentally friendly reaction conditions with excellent regioselectivity. The developed chemistry has been successfully extended for the selective synthesis of C-4 deuterated benzothienopyridines 7u-v and benzofuropyridines 8q-r. The role of the ethanolic proton in the reaction was validated by deuterium-labeling experiments.

Cyclic peptide-selenium nanoparticles as drug transporters.

A cyclic peptide composed of five tryptophan, four arginine, and one cysteine [W5R4C] was synthesized. The peptide was evaluated for generating cyclic peptide-capped selenium nanoparticles (CP-SeNPs) in situ. A physical mixing of the cyclic peptide with SeO3(-2) solution in water generated [W5R4C]-SeNPs via the combination of reducing and capping properties of amino acids in the peptide structure. Transmission electron microscopy (TEM) images showed that [W5R4C]-SeNPs were in the size range of 110-150 nm. Flow cytometry data revealed that a fluorescence-labeled phosphopeptide (F'-PEpYLGLD, where F' = fluorescein) and an anticancer drug (F'-dasatinib) exhibited approximately 25- and 9-times higher cellular uptake in the presence of [W5R4C]-SeNPs than those of F'-PEpYLGLD and dasatinib alone in human leukemia (CCRF-CEM) cells after 2 h of incubation, respectively. Confocal microscopy also exhibited higher cellular delivery of F'-PEpYLGLD and F'-dasatinib in the presence of [W5R4C]-SeNPs compared to the parent fluorescence-labeled drug alone in human ovarian adenocarcinoma (SK-OV-3) cells after 2 h of incubation at 37 °C. The antiproliferative activities of several anticancer drugs doxorubicin, gemcitabine, clofarabine, etoposide, camptothecin, irinotecan, epirubicin, fludarabine, dasatinib, and paclitaxel were improved in the presence of [W5R4C]-SeNPs (50 µM) by 38%, 49%, 36%, 36%, 31%, 30%, 30%, 28%, 24%, and 17%, respectively, after 48 h incubation in SK-OV-3 cells. The results indicate that CP-SeNPs can be potentially used as nanosized delivery tools for negatively charged biomolecules and anticancer drugs.

Base-mediated chemo- and stereoselective addition of 5-aminoindole/tryptamine and histamines onto alkynes.

Transition-metal-free chemo- and stereoselective addition of 5-aminoindole (1a), tryptamine (1b), and histamine (1c) to alkynes 2a-s to synthesize the indolyl/imidazolyl enamines 3a-p, 5a-o, and 6a-e using superbasic solutions of alkali-metal hydroxides in DMSO is described. The addition of N-heterocycles onto alkynes takes places chemoselectively without affecting the 1° amino groups (aromatic and aliphatic) of 5-aminoindole, tryptamine, and histamine. The stereochemistry of the products was found to be dependent upon reaction time; an increase in reaction time leads to the formation of a mixture of E/Z isomers and the thermodynamically stable E addition product. The chemoselective addition of N-heterocycle 1a onto alkyne over thiophenol 7 and phenol 8 is supported by control experiments. Competitive experiments showed that 5-aminoindole was more reactive than tryptamine, and histamine was found to be the least reactive. The present methodology provides an efficient chemoselective method to synthesize a variety of (Z)-enamines of 5-aminoindole, tryptamine, and histamine without affecting the 1° amino group. The presence of the free amino group in enamines could be further used for synthetic elaboration, which proved to be highly advantageous for structural and biological activity assessments.

Synthesis and evaluation of c-Src kinase inhibitory activity of pyridin-2(1H)-one derivatives.

Src kinase, a prototype member of the Src family of kinases (SFKs), is over-expressed in various human tumors, and has become a target for anticancer drug design. In this perspective, a series of eighteen 2-pyridone derivatives were synthesized and evaluated for their c-Src kinase inhibitory activity. Among them, eight compounds exhibited c-Src kinase inhibitory activity with IC50 value of less than 25µM. Compound 1-[2-(dimethylamino)ethyl]-5-(2-hydroxy-4-methoxybenzoyl)pyridin-2(1H)-one (36) exhibited the highest c-Src kinase inhibition with an IC50 value of 12.5µM. Furthermore, the kinase inhibitory activity of compound 36 was studied against EGFR, MAPK and PDK, however no significant activity was observed at the highest tested concentration (300µM). These results provide insights for further optimization of this scaffold for designing the next generation of 2-pyridone derivatives as candidate Src kinase inhibitors.

Broad-spectrum activity of membranolytic cationic macrocyclic peptides against multi-drug resistant bacteria and fungi.

The emergence of multidrug-resistant (MDR) strains causes severe problems in the treatment of microbial infections owing to limited treatment options. Antimicrobial peptides (AMPs) are drawing considerable attention as promising antibiotic alternative candidates to combat MDR bacterial and fungal infections. Herein, we present a series of small amphiphilic membrane-active cyclic peptides composed, in part, of various nongenetically encoded hydrophilic and hydrophobic amino acids. Notably, lead cyclic peptides 3b and 4b showed broad-spectrum activity against drug-resistant Gram-positive (MIC = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria, and fungi (MIC = 3.1-12.5 µg/mL). Furthermore, lead peptides displayed substantial antibiofilm action comparable to standard antibiotics. Hemolysis (HC50 = 230 µg/mL) and cytotoxicity (>70 % cell viability against four different mammalian cells at 100 µg/mL) assay results demonstrated the selective lethal action of 3b against microbes over mammalian cells. A calcein dye leakage experiment substantiated the membranolytic effect of 3b and 4b, which was further confirmed by scanning electron microscopy. The behavior of 3b and 4b in aqueous solution and interaction with phospholipid bilayers were assessed by employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular dynamics (MD) simulations, providing a solid structural basis for understanding their membranolytic action. Moreover, 3b exhibited stability in human blood plasma (t1/2 = 13 h) and demonstrated no signs of resistance development against antibiotic-resistant S. aureus and E. coli. These findings underscore the potential of these newly designed amphiphilic cyclic peptides as promising anti-infective agents, especially against Gram-positive bacteria.

Cyclic peptide-capped gold nanoparticles as drug delivery systems.

A number of cyclic peptides were synthesized and evaluated as simultaneous reducing and capping agents for generation of cyclic peptide-capped gold nanoparticles (CP-AuNPs). Among them, direct dissolution of cyclic peptides containing alternate arginine and tryptophan [WR](n) (n = 3-5) into an aqueous solution of AuCl(4)(-) led to the formation of CP-AuNPs, through the reducing activity of tryptophan residues and attraction of positively charged arginine residues toward chloroaurate anions in the reaction environment. Differential interference contrast microscopy of fluorescence-labeled lamivudine in the presence of [WR](4)-capped AuNPs showed significantly higher cellular delivery of antiviral drug versus that of parent drug alone. Flow cytometry studies also showed that the cellular uptake of fluorescence-labeled lamivudine, emtricitabine, and stavudine was significantly enhanced in human ovarian adenocarcinoma (SK-OV-3) cells in the presence of [WR](4)-AuNPs. For example, fluorescence labeled lamivudine-loaded [WR](4)-AuNPs exhibited approximately 12- and 15-times higher cellular uptake than that of fluorescence labeled lamivudine alone in CCRF-CEM cells and SK-OV-3 cells, respectively. Confocal microscopy revealed that the presence of the [WR](4)-AuNPs enhanced the retention and nuclear localization of doxorubicin in SK-OV-3 cells after 24 h. These data suggest that these complexes can be used as potential noncovalent prodrugs for delivery of antiviral and anticancer agents.

Self-Assembled Surfactant Cyclic Peptide Nanostructures as Stabilizing Agents.

A number of cyclic peptides including [FR]4, [FK]4, [WR]4, [CR]4, [AK]4, and [WK]n (n = 3-5) containing L-amino acids were produced using solid-phase peptide synthesis. We hypothesized that an optimal balance of hydrophobicity and charge could generate self-assembled nanostructures in aqueous solution by intramolecular and/or intermolecular interactions. Among all the designed peptides, [WR]n (n = 3-5) generated self-assembled vesicle-like nanostructures at room temperature as shown by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and/or dynamic light scattering (DLS). This class of peptides represents the first report of surfactant-like cyclic peptides that self-assemble into nanostructures. A plausible mechanistic insight into the self-assembly of [WR]5 was obtained by molecular modeling studies. Modified [WR]5 analogues, such as [WMeR]5, [WR(Me)2]5, [WMeR(Me)2]5, and [WdR]5, exhibited different morphologies to [WR]5 as shown by TEM observations. [WR]5 exhibited a significant stabilizing effect for generated silver nanoparticles and glyceraldehyde-3-phosphate dehydrogenase activity. These studies established a new class of surfactant-like cyclic peptides that self-assembled into nanostructures and could have potential applications for the stabilization of silver nanoparticles and protein biomolecules.

Tandem synthesis of pyrroloacridones via [3 + 2] alkyne annulation/ring-opening with concomitant intramolecular aldol condensation.

An efficient cascade strategy for the direct synthesis of pyrrolo[3,2,1-de]acridones 4a-v, 5a-h from iodo-pyranoquinolines 2a-i by the palladium-catalyzed regioselective [3 + 2] alkyne annulation/ring-opening followed by intramolecular aldol condensation under microwave irradiation is described. The chemistry involves the in situ formation of pyrroloquinolines Y, via palladium-catalyzed selective [3 + 2] annulation of iodopyranoquinolines and internal akynes with ring-opening and successive intramolecular cross-aldol condensation. Both the symmetrical and unsymmetrical internal alkynes were reacted smoothly to provide the desired pyrroloacridones in good yields. This methodology provides the facile conversion of easily accessble iodopyranoquinoline into highly functionalized biologically important pyrroloacridones in a single process.

Silver-catalyzed tandem synthesis of naphthyridines and thienopyridines via three-component reaction.

An efficient approach for the silver-catalyzed regioselective tandem synthesis of highly functionalized 1,2-dihydrobenzo[1,6]naphthyridines 6a-z and 7a-e by the reaction of ortho-alkynylaldehydes 3a-n with amines 4a-d and ketones 5a-c/active methylene compounds 5d-g, under mild reaction conditions, is described. The scope of the developed chemistry was successfully extended for the direct synthesis of 1,2-dihydrobenzo[4,5]thieno[2,3-c]pyridines 8a-e, which is known as the sulfur analogue of ß-carbolines. Naphthyridines 6a-z and thienopyridines 8a-e were obtained via dual activation concept using l-proline as organocatalyst; however, naphthyridines 7a-e were synthesized without using organocatalyst. The reaction shows selective N-C bond formation on the more electrophilic alkynyl carbon, resulting in the regioselective 6-endo-dig-cyclized products. Reactivity behavior of electron-deficient and electron-rich ortho-alkynylaldehydes in the synthesis of naphthyridines and thienopyridine by three-component reaction is supported by the control experiment.

On water: silver-catalyzed domino approach for the synthesis of benzoxazine/oxazine-fused isoquinolines and naphthyridines from o-alkynyl aldehydes.

An operationally simple domino approach for the silver-catalyzed synthesis of oxazine/benzoxazine-fused isoquinolines 5a-q and naphthyridines 6a-v by the reaction of o-alkynyl aldehydes 3a-aa with amines having embedded nucleophiles 4a-d under mild reaction condition in water is described. The reaction shows selective C-N bond formation on the more electrophilic alkynyl carbon resulting in the formation of 6-endo-dig cyclized product. The competitive experiments show the viability of an intramolecular nucleophilic attack over an intermolecular attack of the external nucleophile. This methodology accommodates wide functional group variation, which proves to be useful for structural and biological assessment.

Synthesis and antiproliferative activities of quebecol and its analogs.

Simple and efficient synthesis of quebecol and a number of its analogs was accomplished in five steps. The synthesized compounds were evaluated for antiproliferative activities against human cervix adenocarcinoma (HeLa), human ovarian carcinoma (SK-OV-3), human colon carcinoma (HT-29), and human breast adenocarcinoma (MCF-7) cancer cell lines. Among all the compounds, 7c, 7d, 7f, and 8f exhibited antiproliferative activities against four tested cell lines with inhibition over 80% at 75 µM after 72 h, whereas, compound 7b and 7g were more selective towards MCF-7 cell line. The IC50 values for compounds 7c, 7d, and 7f were 85.1 µM, 78.7 µM, and 80.6 µM against MCF-7 cell line, respectively, showing slightly higher antiproliferative activtiy than the synthesized and isolated quebecol with an IC50 value of 104.2 µM against MCF-7.

Amphiphilic Cell-Penetrating Peptides Containing Arginine and Hydrophobic Residues as Protein Delivery Agents.

The entry of proteins through the cell membrane is challenging, thus limiting their use as potential therapeutics. Seven cell-penetrating peptides, designed in our laboratory, were evaluated for the delivery of proteins. Fmoc solid-phase peptide synthesis was utilized for the synthesis of seven cyclic or hybrid cyclic-linear amphiphilic peptides composed of hydrophobic (tryptophan (W) or 3,3-diphenylalanine (Dip) and positively-charged arginine (R) residues, such as [WR]4, [WR]9, [WWRR]4, [WWRR]5, [(RW)5K](RW)5, [R5K]W7, and [DipR]5. Confocal microscopy was used to screen the peptides as a protein delivery system of model cargo proteins, green and red fluorescein proteins (GFP and RFP). Based on the confocal microscopy results, [WR]9 and [DipR]5 were found to be more efficient among all the peptides and were selected for further studies. [WR]9 (1-10 µM) + protein (GFP and RFP) physical mixture did not show high cytotoxicity (>90% viability) in triple-negative breast cancer cells (MDA-MB-231) after 24 h, while [DipR]5 (1-10 µM) physical mixture with GFP exhibited more than 81% cell viability. Confocal microscopy images revealed internalization of GFP and RFP in MDA-MB-231 cells using [WR]9 (2-10 µM) and [DipR]5 (1-10 µM). Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptake of GFP was concentration-dependent in the presence of [WR]9 in MDA-MB-231 cells after 3 h of incubation at 37 °C. The concentration-dependent uptake of GFP and RFP was also observed in the presence of [DipR5] in SK-OV-3 and MDA-MB-231 cells after 3 h of incubation at 37 °C. FACS analysis indicated that the cellular uptake of GFP in the presence of [WR]9 was partially decreased by methyl-ß-cyclodextrin and nystatin as endocytosis inhibitors after 3 h of incubation in MDA-MB-231 cells, whereas nystatin and chlorpromazine as endocytosis inhibitors slightly reduced the uptake of GFP in the presence of [DipR]5 after 3 h of incubation in MDA-MB-231. [WR]9 was able to deliver therapeutically relevant proteins (Histone H2A) at different concentrations. These results provide insight into the use of amphiphilic cyclic peptides in the delivery of protein-related therapeutics.