Amir Nasrolahi Shirazi

Cell-Penetrating Homochiral Cyclic Peptides as Nuclear-Targeting Molecular Transporters†

The intracellular delivery of biologically active cargos by employing linear cell-penetrating peptides (CPPs) has been previously reported. Conjugation to linear cationic CPPs, such as TAT (trans-acting activator of transcription; a peptide derived from the HIV-1 transactivator protein), 3] penetratin, antennapedia, or oligoarginine, efficiently enhances the cellular uptake through different mechanisms. The cellular uptake and internalization of many CPPs along with the conjugated cargo occurs predominantly by an endocytic pathway that involves macropinocytosis, a caveolae pathway, clathrin-mediated endocytosis, or lipid-raft dependent endocytosis. Endosomal uptake represents a major challenge in targeted intracellular drug delivery since some compounds are trapped in endosomes and cannot reach the biological targets in the cytoplasm or nucleus. Thus, strategies that promote endosomal escape or avoid endosomal routes are required for improving bioavailability. Moreover, the nuclear delivery of cell-impermeable and water-insoluble molecules remains a major challenge. The nucleus is a desirable target because the genetic information of the cell and transcription machinery resides there. To date, most approaches for nuclear delivery of compounds have taken advantage of covalent conjugation, which requires release of the cargo from the conjugate and/or endosomal escape. There is therefore a need to develop alternative stable peptide carriers that avoid endosomal pathways and/or covalent conjugation. Compared to linear peptides that are susceptible to hydrolysis by endogenous peptidases, cyclic peptides are enzymatically more stable. The cell-penetrating properties and application of homochiral l-cyclic peptides in drug delivery remain unexplored. Previous studies on linear CPPs by our research group and others indicated that an optimal balance of positive charge and hydrophobicity is required for interactions with the cell membrane and deep penetration into the lipid bilayer. 4,8–10] Herein, we report the design and evaluation of amphipathic homochiral l-cyclic peptides for potential applications as CPPs and/or as molecular transporters of bioactive compounds. Eleven cyclic peptides, namely [WR]4, [FK]4, [AK]4, [EL]4, [RFEF]2, [EK]4, [ER]4, [FR]4, [RFE]3, [WR]3, and [WR]5 (Scheme 1), which contain l-amino acids, were synthesized by employing 9-fluorenylmethyloxycarbonyl (Fmoc) based peptide chemistry. The selection of the cyclic peptides was based on the presence of hydrophobic residues (e.g., W, F, L) and charged residues (e.g., K, R, E). We hypothesized that an optimal amphipathic cyclic peptide that contains appropriate residues, and that undergoes intermolecular and intramolecular interactions can act as a CPP and/or entrap and deliver a bioactive compound intracellularly. To examine the potential application of the cyclic peptides as molecular transporters, a model experiment was performed with lamivudine (( )-2’,3’-dideoxy-3’-thiacytidine, 3TC). 3TC is a nucleoside reverse transcriptase inhibitor that blocks HIV-1 and hepatitis B virus replication. The efficient cellular uptake of 3TC is critical for effective antiviral activity. To monitor the molecular transport ability of the cyclic peptides, a carboxyfluorescein derivative of 3TC (F-3TC) was synthesized. The cellular uptake of the fluorescently labeled 3TC (F3TC) was examined in the leukemia CCRF-CEM cell line in the presence or absence of cyclic peptides. After 1 h incubation at 37 8C, the cells were treated with trypsin to remove the cell-surface-bound drug. The cellular uptake of F3TC was monitored by fluorescence-activated cell sorting (FACS; Figure 1a) and fluorescence microscopy (Figure 1b). The cyclic peptides did not exhibit any cytotoxicity by using an MTT assay at an experimental concentration of 50 mm in four different cell lines, namely CCRF-CEM, HT-29, MDAMD-468, and SK-OV-3, thus showing consistent results (Figure S12). FACS and fluorescence microscopy showed significantly higher fluorescence signals in the cells treated with F-3TCloaded [WR]4 and [WR]5 compared to those treated with other F-3TC-loaded cyclic peptides and with F-3TC alone, thus suggesting that the uptake of F-3TC is facilitated by [WR]n (n = 4, 5) and is dependent on nature of amino acids. F3TC-loaded [WR]5 exhibited a cellular uptake that was approximately five times higher than that of F-3TC alone (Figure 1a). Phosphopeptides are valuable probes for studying phosphoprotein–protein interactions because these peptides mimic the interactions between the negatively charged phosphate group of phosphoproteins and positively charged amino acids in the binding pockets of a number of proteins. Studying negatively charged phosphopeptides in cellular systems is challenging because these peptides do not readily [*] Dr. D. Mandal, A. Nasrolahi Shirazi, Prof. K. Parang Department of Biomedical and Pharmaceutical Sciences University of Rhode Island 41 Lower College Road, Kingston, RI 02881 (USA) E-mail: kparang@uri.edu

Design and Biological Evaluation of Cell-Penetrating Peptide–Doxorubicin Conjugates as Prodrugs

Doxorubicin (Dox) is a hydrophilic anticancer drug that has short retention time due to the efficient efflux in some cancer cells (e.g., ovarian adenocarcinoma SK-OV-3). Cyclic [W(RW)(4)] and the corresponding linear peptide (RW)(4) were conjugated with Dox through an appropriate linker to afford cyclic [W(RW)(4)]-Dox and linear (RW)(4)-Dox conjugates to enhance the cellular uptake and cellular retention of the parent drug for sustained anticancer activity. Comparative antiproliferative assays between covalent (cyclic [W(RW)(4)]-Dox and linear (RW)(4)-Dox) and the corresponding noncovalent physical mixtures of the peptides and Dox were performed. Cyclic [W(RW)(4)]-Dox inhibited the cell proliferation of human leukemia (CCRF-CEM) (62-73%), ovarian adenocarcinoma (SK-OV-3) (51-74%), colorectal carcinoma (HCT-116) (50-67%), and breast carcinoma (MDA-MB-468) (60-79%) cells at a concentration of 1 μM after 72-120 h of incubation. Cyclic [W(RW)(4)]-Dox exhibited higher antiproliferative activity than linear (RW)(4)-Dox in all cancer cells with the highest activity observed after 72 h. Flow cytometry analysis showed 3.6-fold higher cellular uptake of cyclic [W(RW)(4)]-Dox than Dox alone in SK-OV-3 cells after 24 h incubation. The cellular hydrolysis study showed that 99% of cyclic [W(RW)(4)]-Dox was hydrolyzed intracellularly within 72 h and released Dox. These data suggest that cyclic [W(RW)(4)]-Dox can be used as a potential prodrug for improving the cellular delivery and retention of Dox.

3-Substitued indoles: One-pot synthesis and evaluation of anticancer and Src kinase inhibitory activities

An efficient and economical method was developed for the synthesis of 3-substituted indoles by one-pot three-component coupling reaction of a substituted or unsubstituted benzaldehyde, N-methylaniline, and indole or N-methylindole using Yb(OTf)(3)-SiO(2) as a catalyst. All the synthesized compounds were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), human ovarian adenocarcinoma (SK-OV-3), and c-Src kinase activity. The 4-methylphenyl (4o and 4p) and 4-methoxyphenyl (4q) indole derivatives inhibited the cell proliferation of SK-OV-3 and HT-29 cells by 70-77% at a concentration of 50 μM. The unsubstituted phenyl (4d) and 3-nitrophenyl (4l) derivatives showed the inhibition of c-Src kinase with IC(50) values of 50.6 and 58.3 μM, respectively.

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.

Thiazolyl N-benzyl-substituted acetamide derivatives: Synthesis, Src kinase inhibitory and anticancer activities

KX2-391 (KX-01/Kinex Pharmaceuticals), N-benzyl-2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetamide, is a highly selective Src substrate binding site inhibitor. To understand better the role of pyridine ring and N-benzylsubstitution in KX2-391 and establish the structure-activity relationship, a number of N-benzyl substituted (((2-morpholinoethoxy)phenyl)thiazol-4-yl)acetamide derivatives containing thiazole instead of pyridine were synthesized and evaluated for Src kinase inhibitory activities. The unsubstituted N-benzyl derivative (8a) showed the inhibition of c-Src kinase with GI(50) values of 1.34 μM and 2.30 μM in NIH3T3/c-Src527F and SYF/c-Src527F cells, respectively. All the synthesized compounds were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), breast carcinoma (BT-20), and leukemia (CCRF-CEM) cells. 4-Fluorobenzylthiazolyl derivative 8b exhibited 64-71% inhibition in the cell proliferation of BT-20 and CCRF cells at concentration of 50 μM.

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.

Synthesis and evaluation of novel benzimidazole derivatives as sirtuin inhibitors with antitumor activities.

A total of 15 novel benzimidazole derivatives were designed, synthesized and evaluated for their SIRT1 and SIRT2 inhibitory activity. All compounds showed better inhibition on SIRT2 as compared to SIRT1. Among these, compound 5j displayed the best inhibitory activity for SIRT1 (IC50=58.43μM) as well as for SIRT2 (IC50=45.12μM). Cell cytotoxicity assays also showed that compound 5j possesses good antitumor activity against two different cancer cell lines derived from breast cancer (MCF-7 and MDA-MB-468). A simple structure-activity-relationship (SAR) study of the newly synthesized benzimidazole derivatives was also discussed.

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.

Facile, Regio- and Diastereoselective Synthesis of Spiro-Pyrrolidine and Pyrrolizine Derivatives and Evaluation of Their Antiproliferative Activities

A number of novel spiro-pyrrolidines/pyrrolizines derivatives were synthesized through [3+2]-cycloaddition of azomethine ylides with 3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones 2a–n. Azomethine ylides were generated in situ from the reaction of 1H-indole-2,3-dione (isatin, 3) with N-methylglycine (sarcosine), phenylglycine, or proline. All compounds (50 μM) were evaluated for their antiproliferative activity against human breast carcinoma (MDA-MB-231), leukemia lymphoblastic (CCRF-CEM), and ovarian carcinoma (SK-OV-3) cells. N-α-Phenyl substituted spiro-pyrrolidine derivatives (5a–n) showed higher antiproliferative activity in MDA-MB-231 than other cancer cell lines. Among spiro-pyrrolizines 6a–n, a number of derivatives including 6a–c and 6i–m showed a comparable activity with doxorubicin in all three cell lines. Among all compounds in three classes, 6a, 6b, and 6m, were found to be the most potent derivatives showing 64%, 87%, and 74% antiproliferative activity in MDA-MB-231, SK-OV-3, and CCRF-CEM cells, respectively. Compound 6b showed an IC50 value of 3.6 μM in CCRF-CEM cells. These data suggest the potential antiproliferative activity of spiro-pyrrolidines/pyrrolizines.

Antibacterial activities of amphiphilic cyclic cell-penetrating peptides against multidrug-resistant pathogens.

Multidrug-resistant pathogens have become a major public health concern. There is a great need for the development of novel antibiotics with alternative mechanisms of action for the treatment of life-threatening bacterial infections. Antimicrobial peptides, a major class of antibacterial agents, share amphiphilicity and cationic structural properties with cell-penetrating peptides (CPPs). Herein, several amphiphilic cyclic CPPs and their analogues were synthesized and exhibited potent antibacterial activities against multidrug-resistant pathogens. Among all the peptides, cyclic peptide [R4W4] (1) showed the most potent antibacterial activity against methicillin-resistant Staphylococcus aureus [MRSA, exhibiting a minimal inhibitory concentration (MIC) of 2.67 μg/mL]. Cyclic [R4W4] and the linear counterpart R4W4 exhibited MIC values of 42.8 and 21.7 μg/mL, respectively, against Pseudomonas aeruginosa. In eukaryotic cells, peptide 1 exhibited the expected cell penetrating properties and showed >84% cell viability at a concentration of 15 μM (20.5 μg/mL) in three different human cell lines. Twenty-four hour time-kill studies evaluating [R4W4] with 2 times the MIC in combination with tetracycline demonstrated bactericidal activity at 4 and 8 times the MIC of tetracycline against MRSA (MIC = 0.5 μg/mL) and 2–8 times the MIC against Escherichia coli (MIC = 2 μg/mL). This study suggests that when amphiphilic cyclic CPPs are used in combination with an antibiotic such as tetracycline, they provide significant benefit against multidrug-resistant pathogens when compared with the antibiotic alone.