Cyclic and Linear Peptides Containing Alternate WW and RR Residues as Molecular Cargo Delivery Tools

Cell-impermeable and negatively charged compounds’ cellular uptake across the cell membranes remains challenging. Herein, the synthesis of four linear [(WWRR)2, (WWRR)3, (WWRR)4, and (WWRR)5] and four cyclic ([WWRR]2, [WWRR]3, [WWRR]4, and [WWRR]5) peptides containing alternate two tryptophan (WW) and two arginine (RR) residues and their biological evaluation as molecular transporters are reported. The peptides did not show any significant cytotoxicity in different cell lines (MDA-MB-23, SK-OV-3, and HEK 293) at a concentration of 5 μM and after 3 h of incubation time. The uptake of fluorescence-labeled cargo molecules (F′-GpYEEI, F′-siRNA, and F′-3TC) in the presence of the peptides was monitored in different cell lines (SK-OV-3 and MDA-MB-231) with fluorescence-activated cell sorting. Among all the peptides, [WWRR]5 (C4) showed the highest cellular uptake of cargo molecules, indicating it can act as effective molecular transporter. Confocal microscopy in MDA-MB-231 cells showed the cellular uptake of F′-GpYEEI in the presence of C4 and the intracellular localization of fluorescence-labeled C4 (F′-C4) in the cytosol. The F′-C4 cellular uptake was found to be concentration- and time-dependent, as shown by flow cytometry in MDA-MB-231 cells. Confocal microscopy and flow cytometry of F′-C4 in MDA-MB-231 cells were examined alone and in the presence of different endocytosis inhibitors (chlorpromazine, methyl-β-cyclodextrin, chloroquine, and nystatin). The data showed that the cellular uptake of F′-C4 in the presence of chlorpromazine, chloroquine, and methyl-β-cyclodextrin was reduced but not completely eliminated, indicating that both energy-independent and energy-dependent pathways contributed to the cellular uptake of F′-C4. Similar results were obtained using the confocal microscopy of C4 and F′-GpYEEI in the presence of endocytosis inhibitors (chlorpromazine, methyl-β-cyclodextrin, chloroquine, and nystatin). These data indicate that C4 has the potential to be used as a cell-penetrating peptide and cargo transporter

Efficient Delivery of Cell Impermeable Phosphopeptides by a Cyclic Peptide Amphiphile Containing Tryptophan and Arginine

Phosphopeptides are valuable reagent probes for studying protein–protein and protein–ligand interactions. The cellular delivery of phosphopeptides is challenging because of the presence of the negatively charged phosphate group. The cellular uptake of a number of fluorescent-labeled phosphopeptides, including F′-Gp­YLP­QTV, F′-NEp­YTA­RQ, F′-AE­EEI­YGE­FEA­KK­KK, F′-PEp­YLG­LD, F′-pY­VNV­QN-NH₂, and F′-GpYEEI (F′ = fluorescein), was evaluated in the presence or absence of a [WR]₄, a cyclic peptide containing alternative arginine (R) and tryptophan (W) residues, in human leukemia cells (CCRF-CEM) after 2 h incubation using flow cytometry. [WR]₄ improved significantly the cellular uptake of all phosphopeptides. PEpYLGLD is a sequence that mimics the pTyr1246 of ErbB2 that is responsible for binding to the Chk SH2 domain. The cellular uptake of F′-PEpYLGLD was enhanced dramatically by 27-fold in the presence of [WR]₄ and was found to be time-dependent. Confocal microscopy of a mixture of F′-PEp­YLG­LD and [WR]₄ in live cells exhibited intracellular localization and significantly higher cellular uptake compared to that of F′-PEpYLGLD alone. Transmission electron microscopy (TEM) and isothermal calorimetry (ITC) were used to study the interaction of PEp­YLG­LD and [WR]₄. TEM results showed that the mixture of PEp­YLG­LD and [WR]₄ formed noncircular nanosized structures with width and height of 125 and 60 nm, respectively. ITC binding studies confirmed the interaction between [WR]₄ and PEp­YLG­LD. The binding isotherm curves, derived from sequential binding models, showed an exothermic interaction driven by entropy. These studies suggest that amphiphilic peptide [WR]₄ can be used as a cellular delivery tool of cell-impermeable negatively charged phosphopeptides

Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization

Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, R₅ and R₆. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R₅] and linear peptide dodecanoyl-(R₅) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R₅] and dodecanoyl-[R₆] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F′-GpYEEI in the presence of hexadecanoyl-[R₅] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[R₅] and dodecanoyl-[R₅], respectively. Dodecanoyl-[R₅] enhanced the cellular uptake of the phosphopeptide by 1.4–2.5-fold higher than the corresponding linear peptide dodecanoyl-(R₅) and those of representative CPPs, such as hepta-arginine (CR₇) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids

Peptide Amphiphile Containing Arginine and Fatty Acyl Chains as Molecular Transporters

Peptide amphiphiles (PAs) are promising tools for the intracellular delivery of numerous drugs. PAs are known to be biodegradable systems. Here, four PA derivatives containing arginine and lysine conjugated with fatty acyl groups with different chain lengths, namely, PA1: R-K­(C₁₄)-R, PA2: R-K­(C₁₆)-R, PA3: K­(C₁₄)-R-K­(C₁₄), and PA4: K­(C₁₆)-R-K­(C₁₆), where C₁₆ = palmitic acid and C₁₄ = myristic acid, were synthesized through Fmoc chemistry. Flow cytometry studies showed that, among all synthesized PAs, only K­(C₁₆)-R-K­(C₁₆), PA4 was able to enhance the cellular uptake of a fluorescence-labeled anti-HIV drug 2′,3′-dideoxy-3′-thiacythidine (F′-3TC, F′ = fluorescein) and a biologically important phosphopeptide (F′-PEpYLGLD) in human leukemia cells (CCRF-CEM) after 2 h incubation. For example, the cellular uptake of F′-3TC and F′-PEpYLGLD was enhanced approximately 7.1- and 12.6-fold in the presence of the PA4 compared to those of the drugs alone. Confocal microscopy of F′-3TC and F′-PEpYLGLD loaded PA4 in live cells showed significantly higher intracellular localization than the drug alone in human ovarian cells (SK-OV-3) after 2 h incubation. The high-performance liquid chromatography (HPLC) results showed that loading of Dox by the peptide amphiphile was 56% after 24 h. The loaded Dox was released (34%) within 48 h intracellularly. The circular dichrosim (CD) results exhibited that the secondary structure of the peptide was changed upon interactions with Dox. Mechanistic studies revealed that endocytosis is the major pathway of the internalization. These studies suggest that PAs containing the appropriate sequence of amino acids, chain length, charge, and hydrophobicity can be used as cellular delivery tools for transporting drugs and biomolecules

Molecular modeling of pyrrolo-pyrimidine based analogs as potential FGFR1 inhibitors: a scientific approach for therapeutic drugs

Fibroblast growth factor receptors 1 (FGFR1) is an emerging target for the development of anticancer drugs. Uncontrolled expression of FGFR1 is strongly associated with a number of different types of cancers. Apart from a few FGFR inhibitors, the FGFR family members have not been thoroughly studied to produce clinically effective anticancer drugs. The application of proper computational techniques may aid in understanding the mechanism of protein–ligand complex formation, which may provide a better notion for developing potent FGFR1 inhibitors. In this study, a variety of computational techniques, including 3D-QSAR, flexible docking and MD simulation followed by MMGB/PBSA, H-bonds and distance analysis, have been performed to systematically explore the binding mechanism of pyrrolo-pyrimidine derivatives against FGFR1. The 3D-QSAR model was generated to deduce the structural determinants of FGFR1 inhibition. The high q2 and r2 values for the CoMFA and CoMSIA models indicated that the created 3D-QSAR models could reliably predict the bioactivities of FGFR1 inhibitors. The computed binding free energies (MMGB/PBSA) for the selected compounds were consistent with the ranking of their experimental binding affinities against FGFR1. Furthermore, per-residue energy decomposition analysis revealed that the residues Lys514 in catalytic region, Asn568, Glu571 in solvent accessible portion and Asp641 in DFG motif exhibited a strong tendency to mediate ligand–protein interactions through the hydrogen bonding and Van Der Waals interactions. These findings may benefit researchers in gaining better knowledge of FGFR1 inhibition and may serve as a guideline for the development of novel and highly effective FGFR1 inhibitors. Communicated by Ramaswamy H. Sarma</p