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The cell membrane properties create a significant obstacle in intracellular delivery of cell-impermeable and negatively charged molecules. Herein, we report the synthesis and biological evaluation of a novel series of hybrid cyclic-linear peptides containing alternative positive and hydrophobic amino acids on the ring and side chain [(RW)5]K(RW)X (X = 1-5) to compare their molecular transporter efficiency. The peptides were synthesized through Fmoc solid-phase peptide synthesis. In vitro cytotoxicity of the peptides showed that the peptides did not exhibit any significant cytotoxicity at the concentration of 10 μM in human leukemia carcinoma cell line (CCRF-CEM), human ovarian adenocarcinoma cells (SK-OV-3), human epithelial embryonic kidney healthy (HEK-293), and human epithelial mammary gland adenocarcinoma cells (MDA-MB-231) after 3 h incubation. The cellular uptake of a fluorescence-labeled phosphopeptide (F'-GpYEEI) and anti-human immunodeficiency virus (HIV) drugs (lamivudine (F'-3TC), emtricitabine (F'-FTC), Stavudine (F'-d4T)), where F' is carboxyfluorescein, was measured in the presence of the peptides in CCRF-CEM and SK-OV-3 cells. Among all peptides, [(RW)5K](RW)5 (10 μM) was the most efficient transporter that improved the cellular uptake of F'-GpYEEI (2 μM) by 18- and 11-fold in CCRF-CEM and SK-OV-3, respectively, compared with F'-GpYEEI alone. Fluorescence-activated cell sorting (FACS) analysis results indicated that the cellular uptake of fluorescence-labeled peptide (F'-[(RW)5K](RW)5) was only partially inhibited by chlorpromazine as an endocytosis inhibitor after 3 h incubation in MDA-MB-231 cells. These data suggest the potential of this series of hybrid cyclic-linear peptides as cell-penetrating peptides and molecular transporters.

We designed a library of 24 cyclic peptides containing arginine (R) and tryptophan (W) residues in a sequential manner [R

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The activity of nucleoside and nucleotide analogs as antiviral agents requires phosphorylation by endogenous enzymes. Phosphate-substituted analogs have low bioavailability due to the presence of ionizable negatively-charged groups. To circumvent these limitations, several prodrug approaches have been proposed. Herein, we hypothesized that the conjugation or combination of the lipophilic amide bond with nucleotide-based tenofovir (TFV) (1) could improve the anti-HIV activity. During the current study, the hydroxyl group of phosphonates in TFV was conjugated with the amino group of L-alanine, L-leucine, L-valine, and glycine amino acids and other long fatty ester hydrocarbon chains to synthesize 43 derivatives. Several classes of derivatives were synthesized. The synthesized compounds were characterized by 1H NMR, IR, UV, and mass spectrometry. In addition, several of the synthesized compounds were evaluated as racemic mixtures for anti-HIV activity in vitro in a single round infection assay using TZM-bl cells at 100 ng/mL. TFV (1) was used as a positive control and inhibited HIV infection by 35%. Among all the evaluated compounds, the disubstituted heptanolyl ester alanine phosphonamidate with naphthol oleate (69), pentanolyl ester alanine phosphonamidate with phenol oleate (62), and butanolyl ester alanine phosphonamidate with naphthol oleate (87) ester conjugates of TFV were more potent than parent drug TFV with 79.0%, 76.5%, 71.5% inhibition, respectively, at 100 ng/mL. Furthermore, two fatty acyl amide conjugates of tenofovir alafenamide (TAF) were synthesized and evaluated for comparative studies with TAF and TFV conjugates. Tetradecanoyl TAF conjugate 95 inhibited HIV infection by 99.6% at 100 ng/mL and showed comparable activity to TAF (97–99% inhibition) at 10–100 ng/mL but was more potent than TAF when compared at molar concentration.

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

Human malignant melanoma exhibits imbalances in redox status, leading to activation of many redox-sensitive signaling pathways. APE/Ref-1 is a multifunctional protein that serves as a redox chaperone that regulates many nuclear transcription factors and is an important mechanism in cancer cell survival of oxidative stress. Previous studies showed that APE/Ref-1 is a potential druggable target for melanoma therapy. In this study, we synthesized a novel APE/Ref-1 inhibitor, bis-cinnamoyl-1,12-dodecamethylenediamine (2). In a xenograft mouse model, compound 2 treatment (5 mg/kg) significantly inhibited tumor growth compared to the control group, with no significant systemic toxicity observed. We further synthesized compound 2 analogs to determine the structure-activity relationship based on their anti-melanoma activities. Among those, 4-hydroxyphenyl derivative (11) exhibited potent anti-melanoma activities and improved water solubility compared to its parental compound 2. The IC50 of compound 11 was found to be less than 0.1 μM. Compared to other known APE/Ref-1 inhibitors, compound 11 exhibited increased potency in inhibiting melanoma proliferation. As determined by luciferase reporter analyses, compound 2 was shown to effectively inhibit H2O2-activated AP-1 transcription activities. Targeting APE/Ref-1-mediated signaling using pharmaceutical inhibitors is a novel and effective strategy for melanoma treatment with potentially high impact.

Several drug molecules have shown low bioavailability and pharmacokinetic profile due to metabolism by enzymes, excretion by the renal system, or due to other physiochemical properties of drug molecules. These problems have resulted in the loss of efficacy and the gain of side effects associated with drug molecules. PEGylation is one of the strategies to overcome these pharmacokinetic issues and has been successful in the clinic. Cell-penetrating Peptides (CPPs) help to deliver molecules across biological membranes and could be used to deliver cargo selectively to the intracellular site or to the drug target. Hence CPPs could be used to improve the efficacy and selectivity of the drug. However, due to the peptidic nature of CPPs, they have a low pharmacokinetic profile. Using PEGylation and CPPs together as a component of a drug delivery system, the and efficacy of drug molecules could be improved. The other important pharmacokinetic properties such as short half-life, solubility, stability, absorption, metabolism, and elimination could be also improved. Here in this review, we summarized PEGylated CPPs or PEGylation based formulations for CPPs used in a drug delivery system for several biomedical applications until August 2019.

A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5–10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F’-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F’-[K(DipR)5]) alone and F’-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F’-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F’-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F’-[K(DipR)5] alone or F’-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

We report the synthesis and antibacterial activities of a series of amphiphilic membrane-active peptides composed, in part, of various nongenetically coded hydrophobic amino acids. The lead cyclic peptides

Doxorubicin (Dox) is an anthracycline chemotherapeutic agent used to treat breast, leukemia, and lymphoma malignancies. However, cardiotoxicity and inherent acquired resistance are major drawbacks, limiting its clinical application. We have previously shown that cyclic peptide [WR]9 containing alternate tryptophan (W) and arginine (R) residues acts as an efficient molecular transporter. An amphiphilic cyclic peptide containing a lysine (K) residue and alternative W and R was conjugated through a free side chain amino group with Dox via a glutarate linker to afford [(WR)8WKβA]-Dox conjugate. Antiproliferative assays were performed in different cancer cell lines using the conjugate and the corresponding physical mixture of the peptide and Dox to evaluate the effectiveness of synthesized conjugate compared to the parent drug alone. [(WR)8WKβA]-Dox conjugate showed higher antiproliferative activity at 10 µM and 5 µM than Dox alone at 5 μM. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and the triple-negative breast cancer cells MDA-MB-231 and MCF-7 by 71% and 77%, respectively, at a concentration of 5 μM after 72 h of incubation. In contrast, Dox inhibited the proliferation of SK-OV-3, MDA-MB-231, and MCF-7 by 35%, 63%, and 57%, respectively. Furthermore, [(WR)8WKβA]-Dox conjugate (5 µM) inhibited the cell viability of Dox-resistant cells (MES-SA/MX2) by 92%, while the viability of cells incubated with free Dox was only 15% at 5 μM. Confocal microscopy images confirmed the ability of both Dox conjugate and the physical mixture of the peptide with the drug to deliver Dox through an endocytosis-independent pathway, as the uptake was not inhibited in the presence of endocytosis inhibitors. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. Half-life (t1/2) for [(WR)8WKβA]-Dox conjugate was (∼6 h), and more than 80% of the conjugate was degraded at 12 h. The release of free Dox was assessed intracellularly using the CCRF-CEM cell line. The experiment demonstrated that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h. These data confirm the ability of the cyclic cell-penetrating peptide containing tryptophan and arginine residues as an efficient tool for delivery of Dox and for overcoming resistance to it.

Cyclic dipeptides, also know as diketopiperazines (DKP), the simplest cyclic forms of peptides widespread in nature, are unsurpassed in their structural and bio-functional diversity. DKPs, especially those containing proline, due to their unique features such as, inter alia, extra-rigid conformation, high resistance to enzyme degradation, increased cell permeability, and expandable ability to bind a diverse of targets with better affinity, have emerged in the last years as biologically pre-validated platforms for the drug discovery. Recent advances have revealed their enormous potential in the development of next-generation theranostics, smart delivery systems, and biomaterials. Here, we present an updated review on the biological and structural profile of these appealing biomolecules, with a particular emphasis on those with anticancer properties, since cancers are the main cause of death all over the world. Additionally, we provide a consideration on supramolecular structuring and synthons, based on the proline-based DKP privileged scaffold, for inspiration in the design of compound libraries in search of ideal ligands, innovative self-assembled nanomaterials, and bio-functional architectures.

CGKRK is a well-known tumor homing peptide with significant specificity for many types of cancer tissues. Herein, we describe the synthesis of a novel drug delivery system based on dextran decorated with myristoyl-ECGKRK peptide. The myristoylated peptide was synthesized and conjugated to dextran via an ester bond followed by purification. FT-IR and NMR confirmed the success of the conjugation reaction, while the surface morphology examination revealed that the conjugate has a characteristic porous network-like structure. Dynamic-light scattering measurements indicated the ability of the conjugate to self-assemble into nanoparticles with an average size of 248 ± 6.33 nm, and zeta potential of 10.7 mV. The cytotoxicity profiles for the peptide, dextran (Dex0), and dextran-peptide conjugate (Dex1) were evaluated against triple-negative breast cancer cells (MDA-MB-231), breast cancer cells (MCF-7), and human embryonic normal kidney cells (HEK-293). The results revealed that myristoyl-ECGKRK was noncytotoxic on the two different breast cancer cell lines up to 50 μM, but the cell viability was minimally reduced to 85% at 50 μm in HEK-293 cells. Similarly, Dex0 showed a neglected cytotoxicity profile at all tested concentrations. The Dex1 was not toxic to the cells up to a concentration of 8.3 mg/mL.

Doxorubicin (Dox) is used for breast cancer, leukemia, and lymphoma treatment as an effective chemotherapeutic agent. However, Dox use is restricted due to inherent and acquired resistance and an 8-fold increase in the risk of potentially fatal cardiotoxicity. Hybrid cyclic-linear peptide [R5K]W7A and linear peptide R5KW7A were conjugated with Dox through a glutarate linker to afford [R5K]W7A-Dox and R5KW7A-Dox conjugates to generate Dox derivatives. Alternatively, [R5K]W7C was conjugated with Dox via a disulfide linker to generate [R5K]W7C-S-S-Dox conjugate, where S-S is a disulfide bond. Comparative antiproliferative assays between conjugates [R5K]W7A-Dox, [R5K]W7C-S-S-Dox, linear R5KW7A-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [R5K]W7A-Dox conjugate was 2-fold more efficient than R5KW7A-Dox, and [R5K]W7C-S-S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [R5K]W7A-Dox conjugate was selected for further studies and inhibited the cell viability of CCRF-CEM (84%), ovarian adenocarcinoma (SK-OV-3, 39%), and gastric carcinoma (AGS, 73%) at a concentration of 5 μM after 72 h of incubation, which was comparable to Dox (5 μM) efficacy (CCRF-CEM (85%), SK-OV-3 (33%), and AGS (87%)). While [R5K]W7A-Dox had a significant effect on the viability of cancer cells, it exhibited minimal cytotoxicity to normal kidney (LLC-PK1, 5-7%) and heart cells (H9C2

Under-expression or over-expression of protein kinases has been shown to be associated with unregulated cell signal transduction in cancer cells. Therefore, there is a 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, [R(6)K]W(6) and [R(5)K]W(7), 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 (IC(50)= 0.21 μM) than [WR](5), [WR](6), [WR](7), and [WR](8) with IC(50) values of 0.81, 0.57, 0.35, and 0.33 μM, respectively, against Src kinase as determined by a radioactive assay using [γ-(33)P]ATP. Consistent with the result above, [WR](9) inhibited other protein kinases such as Abl kinase activity with an IC(50) value of 0.35 μM, showing 2.2-fold higher inhibition than [WR](5) (IC(50)= 0.79 μM). [WR](9) also inhibited PKCa kinase activity with an IC(50) value of 2.86 μM, approximately 3-fold higher inhibition than [WR](5) (IC(50)=8.52 μM). A similar pattern was observed against Braf, c-Src, Cdk2/Cyclin A1, and Lck. [WR](9) exhibited IC(50) values of

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1–reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493–0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

RNAi is a biological process that utilizes small interfering RNA (siRNA) to prevent the translation of mRNA to protein. This mechanism could be beneficial in preventing the overexpression of proteins in cancer. However, the cellular delivery of siRNA has proven to be challenging due to its inherent negative charge and relative instability. Here, we designed a multicomponent delivery system composed of a specifically designed peptide (linear or cyclic fatty acyl peptide conjugates and hybrid cyclic/linear peptides) and several lipids (DOTAP, DOPE, cholesterol, and phosphatidylcholine) to form a nanoparticle, which we have termed as peptide lipid-associated nucleic acids (PLANAs). Five formulations were prepared (a formulation with no peptide, which was named lipid-associated nucleic acid or LANA, and PLANA formulations A-D) using a mini extruder to form uniform nanoparticles around 100 nm in size with a slightly positive charge (less than +10 mv). Formulations were evaluated for peptide incorporation, siRNA encapsulation efficiency, release profile, toxicity, cellular uptake, and protein silencing. Our experiments showed effective encapsulation of siRNA (>95%), a controlled release profile, and negligible toxicity in formulations that did not contain a positively charged lipid. The results also revealed that PLANAs C and D exhibited optimum cellular uptake (with 80-90% siRNA-positive cells for most of the formulations). PLANA D formulation was selected to silence two model proteins (Src and RPS6KA5) in the triple-negative human breast cancer cell line MDA-MB-231, with promising silencing efficiency, which diminished the expression of RPS6KA5 and Src to approximately 29 and 38% compared to naive cells, respectively. Many approaches have been investigated for safe and efficient delivery of nucleic acids in the last 20 years; however, many have failed due to the multifaceted challenges to overcome. Our results show a promising potential for a multicomponent design that incorporates different components for a variety of delivery tasks, which warrants further investigation of PLANAs in vivo.

Peptides are fragments of proteins that carry out biological functions. They act as signaling entities via all domains of life and interfere with protein-protein interactions, which are indispensable in bio-processes. Short peptides include fundamental molecular information for a prelude to the symphony of life. They have aroused considerable interest due to their unique features and great promise in innovative bio-therapies. This work focusing on the current state-of-the-art short peptide-based therapeutical developments is the first global review written by researchers from all continents, as a celebration of 100 years of peptide therapeutics since the commencement of insulin therapy in the 1920s. Peptide “drugs” initially played only the role of hormone analogs to balance disorders. Nowadays, they achieve numerous biomedical tasks, can cross membranes, or reach intracellular targets. The role of peptides in bio-processes can hardly be mimicked by other chemical substances. The article is divided into independent sections, which are related to either the progress in short peptide-based theranostics or the problems posing challenge to bio-medicine. In particular, the SWOT analysis of short peptides, their relevance in therapies of diverse diseases, improvements in (bio)synthesis platforms, advanced nano-supramolecular technologies, aptamers, altered peptide ligands and in silico methodologies to overcome peptide limitations, modern smart bio-functional materials, vaccines, and drug/gene-targeted delivery systems are discussed.

We have previously reported cyclic cell-penetrating peptides [WR]5 and [WR]4 as molecular transporters. To optimize further the utility of our developed peptides for targeted therapy in cancer cells using the redox condition, we designed a new generation of peptides and evaluated their cytotoxicity as well as uptake behavior against different cancer cell lines. Thus, cyclic [C(WR)xC] and linear counterparts (C(WR)xC), where x = 4&ndash, 5, were synthesized using Fmoc/tBu solid-phase peptide synthesis, purified, and characterized. The compounds did not show any significant cytotoxicity (at 25 &micro, M) against ovarian (SK-OV-3), leukemia (CCRF-CEM), gastric adenocarcinoma (CRL-1739), breast carcinoma (MDA-MB-231), and normal kidney (LLCPK) cells after 24 and 72 h incubation. Both cyclic [C(WR)5C] and linear (C(WR)5C) demonstrated comparable molecular transporter properties versus [WR]5 in the delivery of a phosphopeptide (F&prime, GpYEEI) in CCRF-CEM cells. The uptake of F&prime, GpYEEI in the presence of 1,4-dithiothreitol (DTT) as the reducing agent was significantly improved in case of l(C(WR)5C), while it was not changed by [C(WR)5C]. Fluorescence microscopy also demonstrated a significant uptake of F&prime, GpYEEI in the presence of l(C(WR)5C). Cyclic [C(WR)5C] improved the uptake of the fluorescent-labeled anti-HIV drugs F&prime, d4T, F&prime, 3TC, and F&prime, FTC by 3.0&ndash, 4.9-fold. These data indicate that both [C(WR)5C] and linear (C(WR)5C) peptides can act as molecular transporters.

N1-(&alpha, &beta, Alkene)-substituted phenylpyrazolopyrimidine derivatives with acetyl and functionalized phenyl groups at &alpha, and &beta, positions, respectively, were synthesized by the reaction of 3-phenylpyrazolopyrimidine (PhPP) with bromoacetone, followed by a chalcone reaction with differently substituted aromatic aldehydes. The Src kinase enzyme assay revealed modest inhibitory activity (half maximal inhibitory concentration, IC50 = 21.7&ndash, 192.1 &micro, M) by a number of PhPP derivatives. Antiproliferative activity of the compounds was evaluated on human leukemia (CCRF-CEM), human ovarian adenocarcinoma (SK-OV-3), breast carcinoma (MDA-MB-231), and colon adenocarcinoma (HT-29) cells in vitro. 4-Chlorophenyl carbo-enyl substituted 3-phenylpyrazolopyrimidine (10) inhibited the cell proliferation of HT-29 and SK-OV-3 by 90% and 79%, respectively, at a concentration of 50 &micro, M after 96 h incubation. The compound showed modest inhibitory activity against c-Src (IC50 = 60.4 &micro, M), Btk (IC50 = 90.5 &micro, M), and Lck (IC50 = 110 &micro, M), while it showed no activity against Abl1, Akt1, Alk, Braf, Cdk2, and PKCa. In combination with target selection and kinase profiling assay, extensive theoretical studies were carried out to explore the selectivity behavior of compound 10. Specific interactions were also explored by examining the changing trends of interactions of tyrosine kinases with the phenylpyrazolopyrimidine derivative. The results showed good agreement with the experimental selectivity pattern among c-Src, Btk, and Lck.

The Alzheimer’s brain is affected by multiple pathophysiological processes, which include a unique, organ-specific form of insulin resistance that begins early in its course. An additional complexity arises from the four-fold risk of Alzheimer’s Disease (AD) in type 2 diabetics, however there is no definitive proof of causation. Several strategies to improve brain insulin signaling have been proposed and some have been clinically tested. We report findings on a small allosteric molecule that reverses several indices of insulin insensitivity in both cell culture andin vitromodels of AD that emphasize the intracellular accumulation of β-amyloid (Aβi). PS48, a chlorophenyl pentenoic acid, is an allosteric activator of PDK-1, which is an Akt-kinase in the insulin/PI3K pathway. PS48 was active at 10 nM to 1 μM in restoring normal insulin-dependent Akt activation and in mitigating Aβi peptide toxicity. Synaptic plasticity (LTP) in prefrontal cortical slices from normal rat exposed to Aβ oligomers also benefited from PS48. During these experiments, neither overstimulation of PI3K/Akt signaling nor toxic effects on cells was observed. Another neurotoxicity model producing insulin insensitivity, utilizing palmitic acid, also responded to PS48 treatment, thus validating the target and indicating that its therapeutic potential may extend outside of β-amyloid reliance. The describedin vitroandcell based-in vitrocoupled enzymatic assay systems proved suitable platforms to screen a preliminary library of new analogs.

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3′ position. Monofatty acyl conjugates, 3′-O-tetradecanoyl (4a) (IC50(VeroE6) = 2.3 μM; IC50(Calu3) = 0.24 μM), 3′-O-4-oxatetradodecanoyl (4b) (IC50(VeroE6) = 2.0 μM; IC50(Calu3) = 0.18 μM), and 3′-O-(12-ethylthiododecanoyl) (4e) (IC50(VeroE6) = 2.4 μM; IC50(Calu3) = 0.25 μM) derivatives exhibited less activity than RDV (IC50(VeroE6) = 0.85 μM; IC50(Calu3) = 0.06 μM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV., Graphical abstract Image 1

A series of 11 unsymmetrical dicarboxylate conjugates of dinucleoside reverse transcriptase inhibitors were synthesized. Three dicarboxylic acids, succinic acid, suberic acid and 1,14-tetradecandioc acid, were diesterified with either 3′-azido-2′,3′-dideoxythymidine (AZT), 3′-fluoro-2′,3′-dideoxythymidine (FLT), 2′,3′-dideoxy-3′-thiacytidine (3TC), or 5-fluoro-2′,3′-dideoxy-3′-thiacytidine (FTC). The anti-HIV activity of synthesized compounds was evaluated against HIV-1 X4 (IIIB) and R5 (BaL) viral strains in single-round infection assays. Results indicated that the tetradecandioate esters of nucleosides were more active against HIV than the corresponding parent nucleosides and nucleoside conjugates. The tetradecandioate conjugate of FLT and FTC (5) was found to be the most potent compounds with EC50 values of 47 and 75 nM against X4 and R5 HIV-1 strains, respectively, while the EC50 values for the parent analogs, FLT and FTC, ranged from 700 to 3300 nM.

Cyclic cell-penetrating peptides are relatively a newer class of peptides that have a huge potential for the intracellular delivery of therapeutic agents aimed at treating challenging ailments like multidrug-resistant bacterial diseases, cancer, and HIV infection. Cell-penetrating peptides (CPPs) have been extensively explored as intracellular delivery vehicles; however, they have some inherent limitations like poor stability, endosomal entrapment, toxicity, and suboptimal cell penetration. Owing to their favorable properties that avoid these limitations, cyclic CPPs can provide a good alternative to linear CPPs. Several Reviews have been published in the past decade that cover CPPs and cyclic peptides independently. To the best of our knowledge, this is one of the first Reviews that covers cyclic CPPs comprehensively in the light of studies published so far. In this Review, we have detailed examples of cyclic CPPs, their structures, and cyclization strategies followed by a detailed account of their advantages over their linear counterparts. A hot area in cyclic CPPs is the exploration of cell-penetration mechanisms; this Review highlights this topic in detail. Finally, we will review the applications of cyclic CPPs, followed by conclusions and future prospects.

Prostate cancer (PCa) is the most common malignancy in men and is the leading cause of cancer-related male mortality. A disulfide cyclic peptide ligand [CTVRTSADC] 1 has been previously found to target extra domain B of fibronectin (EDB-FN) in the extracellular matrix that can differentiate aggressive PCa from benign prostatic hyperplasia. We synthesized and optimized the stability of ligand 1 by amide cyclization to obtain [KTVRTSADE] 8 using Fmoc/tBu solid-phase chemistry. Optimized targeting ligand 8 was found to be stable in phosphate buffered saline (PBS, pH 6.5, 7.0, and 7.5) and under redox conditions, with a half-life longer than 8 h. Confocal microscopy studies demonstrated increased binding of ligand 8 to EDB-FN compared to ligand 1. Therefore, we hypothesized that the EDB-FN targeted peptides (1 and 8) conjugated with an anticancer drug via a hydrolyzable linker would provide selective cytotoxicity to the cancer cells. To test our hypothesis, we selected both the normal prostate cell line, RWPE-1, and the cancerous prostate cell lines, PC3, DU-145, LNCaP, and C4-2, to evaluate the anticancer activity of synthesized peptide&ndash, drug conjugates. Docetaxel (Doce) and doxorubicin (Dox) were used as anticancer drugs. Dox conjugate 13 containing disulfide linkage showed comparable cytotoxicity versus Dox after 72 h incubation in all the cancer cell lines, whereas it was found to be less cytotoxic on RWPE-1, suggesting that it can act as a Dox prodrug. Doce conjugate 14 was found to be less cytotoxic in all the cell lines as compared to drug alone.

Cell-penetrating peptide [WR]5 has been previously shown to be an efficient molecular transporter for various hydrophilic and hydrophobic molecules. The peptide was synthesized using Fmoc/tBu solid-phase chemistry, and one arginine was replaced with one lysine to enable the conjugation with the anticancer drugs. Paclitaxel (PTX) was functionalized with an esterification reaction at the C2&prime, hydroxyl group of PTX with glutaric anhydride and conjugated with the cyclic peptide [W(WR)4K(&beta, Ala)] in DMF to obtain the peptide-drug conjugate PTX1. Furthermore, camptothecin (CPT) was modified at the C(20)-hydroxyl group through the reaction with triphosgene. Then, it was conjugated with two functionalized cyclic peptides through a formyl linker affording two different conjugates, namely CPT1 and CPT2. All the conjugates showed better water solubility as compared to the parent drug. The cytotoxicity assay of the drugs and their conjugates with the peptides were evaluated in the human breast cancer MCF-7 cell line. PTX inhibited cell proliferation by 39% while the PTX-peptide conjugate inhibited the proliferation by ~18% after 72 h incubation. On the other hand, CPT, CPT1, and CPT2 reduced the cell proliferation by 68%, 39%, and 62%, respectively, in the MCF-7 cell lines at 5 &micro, M concentration after 72 h incubation.

Targeting cancer cells using integrin receptor is one of the promising targeting strategies in drug delivery. In this study, we conjugated an integrin-binding ligand (GGRGDSK) peptide to chitosan oligosaccharide (COS) using sulfo-SMCC as a bifunctional linker to afford COS-SMCC-GGRGDSK. The conjugated polymer was characterized by FT-IR, (1)H NMR, (13)C NMR, and SEM. COS-SMCC-GGRGDSK did not show cytotoxicity up to a concentration of 1mg/mL in the human leukemia cell line (CCRF-CEM). The conjugate was evaluated for its ability to enhance the cellular uptake of a cell-impermeable cargo (e.g., F'-G(pY)EEI phosphopeptide) in CCRF-CEM, and human ovarian carcinoma (SK-OV-3) cancer cell lines. Additionally, RGD modified and unmodified COS polymers were used to prepare nanoparticles by ionic gelation and showed particle size ranging from 187 to 338nm, and zeta potential of 12.2-18.3mV using dynamic light scattering. The efficiency of COS-NPs and COS-SMCC-RGDSK NPs was assayed for translocation of two synthetic cytotoxic agents ((2-(2-aminoethylamino)-4-(4-chlorophenyl)-6-(1H-indol-3-yl) nicotinonitrile (ACIN), and 2-(2-aminoethylamino)-6-(1H-indol-3-yl)-4-(4-methoxyphenyl)-nicotinonitrile (AMIN)) into CCRF-CEM and human prostate (DU-145) cancer cell lines. The results showed a dramatic reduction in the cell viability on their treatment with RGD targeted COS NPs in comparison to paclitaxel (PTX), free drug, and drug-loaded COS NPs.

Due to the low water solubility and hydrophobic nature of curcumin, an efficient cellular uptake is critical for its biological activity. We have previously developed a number of homochiral L-cyclic peptides containing arginine and tryptophan as cell-penetrating peptides. Among the synthesized peptides, [WR]5 containing five arginine and five tryptophan residues was found to be the most efficient one. Here, we have compared the application of [WR]5 to improve the intracellular uptake of curcumin by using both peptide-curcumin conjugate and physical mixture (peptide + curcumin) strategies. Flow cytometry results showed that the intracellular uptake of curcumin (50 μM) was enhanced through the physical mixing with [WR]5 by 5.7 folds compared to that of curcumin alone in human leukemia (CCRFCEM) cells after 3 h. When [WR]5 was conjugated with curcumin, the intracellular uptake was enhanced by 4 fold. These data suggest that the physical mixture can work more efficiently in enhancing the cellular delivery of curcumin. Furthermore, the antiproliferative activity of curcumin was enhanced by 20% and ∼13% through the physical mixture and the conjugate, respectively, in CCRF-CEM cells after 72 h.

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.

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.

Myocardial toxicity and drug resistance caused by drug efflux are major limitations of doxorubicin (Dox)-based chemotherapy. Dox structure modification could be used to develop conjugates with an improved biological profile, such as antiproliferative activity and higher cellular retention. Thus, Dox thiol conjugates, Dox thiol (Dox-SH), thiol-reactive Dox-SS-pyridine (SS = disulfide), and a Dox-SS-cell-penetrating cyclic peptide, Dox-SS-[C(WR)(4)K], were synthesized. Dox was reacted with Trauťs reagent to generate Dox-SH. The thiol group was activated by the reaction with dithiodipyridine to afford the corresponding Dox-SS-Pyridine (Dox-SS-Pyr). A cyclic cell-penetrating peptide containing a cysteine residue [C(WR)(4)K] was prepared using Fmoc solid-phase strategy. Dox-SS-Py was reacted with the free sulfhydryl of cysteine in [C(WR)(4)K] to generate Dox-SS-[C(WR)(4)K] as a Dox-cyclic peptide conjugate. Cytotoxicity of the compounds was examined in human embryonic kidney (HEK-293), human ovarian cancer (SKOV-3), human fibrosarcoma (HT-1080), and human leukemia (CCRF-CEM) cells. Dox-SH and Dox-SS-pyridine were found to have significantly higher or comparable cytotoxicity when compared to Dox in HEK-293, HT-1080, and CCRF-CEM cells after 24 h and 72 incubation, presumably because of higher activity and retention of the compounds in these cells. Furthermore, Dox-SS-[C(WR)(4)K] showed significantly higher cytotoxic activity in HEK-293, HT-1080, and SKOV-3 cells when compared with Dox after 72 h incubation. Dox-SS-Pyr exhibited higher cellular uptake than Dox-SS-[C(WR)(4)K] in HT-1080 and HEK-293 cells as shown by flow cytometry. Fluorescence microscopy exhibited that Dox-SS-Pyr, Dox-SH, and Dox-SS-[C(WR)(4)K] localized in the nucleus as shown in four cell lines, HT-1080, SKOV-3, MDA-MB-468, and MCF-7. Of note, Dox-SS-[C(WR)(4)K] was significantly less toxic in mouse myoblast cells compared to Dox at the same concentration. Further mechanistic study demonstrated that the level of intracellular reactive oxygen species (ROS) in myoblast cells exposed to Dox-SS-[C(WR)(4)K] was reduced in comparison of Dox when co-treated with FeCl(2). These data indicate that Dox-SH, Dox-SS-Pyr, and Dox-SS-[C(WR)(4)K] have the potential to be further examined as Dox alternatives and anticancer agents.

Linear (HR)n and cyclic [HR]n peptides (n = 4,5) containing alternate arginine and histidine residues were synthesized. The peptides showed 0&ndash, 15% cytotoxicity at 5&ndash, 100 &micro, M in human ovarian adenocarcinoma (SK-OV-3) cells while they exhibited 0&ndash, 12% toxicity in human leukemia cancer cell line (CCRF-CEM). Among all peptides, cyclic [HR]4 peptide was able to improve the delivery of a cell impermeable fluorescence-labeled phosphopeptide by two-fold. Fatty acids of different alkyl chain length were attached at the N-terminal of the linear peptide (HR)4 to improve the molecular transporter property. Addition of fatty acyl chains was expected to help with the permeation of the peptides through the cell membrane. Thus, we synthesized seven fatty acyl derivatives of the linear (HR)4 peptide. The peptides were synthesized using Fmoc/tBu solid phase peptide chemistry, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption/ionization (MALDI) spectrometry. The fatty acyl peptides containing C8, C12, C14, and C18 alkyl chain did not show cytotoxicity on SK-OV-3 or CCRF-CEM cell lines up to 50 &micro, M concentration, however, at higher concentration (100 &micro, M), they showed mild cytotoxicity. For example, C16-(HR)4 was also found to reduce the proliferation of SK-OV-3 cells by 11% at 50 &micro, M and C20-(HR)4 showed mild toxicity at 10 &micro, M, reducing the proliferation of SK-OV-3 cells by 21%. Increase in the length of alkyl chain showed cytotoxicity to the cell lines. C20-(HR)4 peptide showed better efficiency in translocation of F&prime, GpYEEI to SK-OV-3 than the phosphopeptide alone. Further investigation of C20-(HR)4 peptide efficacy showed that the peptide could deliver doxorubicin and epirubicin into SK-OV-3 and also improved the drug antiproliferative ability. These studies provided insights into understanding the structural requirements for optimal cellular delivery of the fatty acyl-(HR)4 peptide conjugates.

Tumor-targeted carriers provide efficient delivery of chemotherapeutic agents to tumor tissue. CGKRK is one of the well-known tumor targeting peptides with significant specificity for angiogenic blood vessels and tumor cells. Here, we designed fatty acyl conjugated CGKRK peptides, based on the hypothesis that hydrophobically-modified CGKRK peptide could enhance cellular permeation and delivery of siRNA targeted to tumor cells for effective silencing of selected proteins. We synthesized six fatty acyl-peptide conjugates, using a diverse chain of saturated and unsaturated fatty acids to study the efficiency of this approach. At peptide:siRNA weight/weight ratio of 10:1 (N/P ≈ 13.6), almost all the peptides showed complete binding with siRNA, and at a w/w ratio of 20:1 (N/P ≈ 27.3), complete protection of siRNA from early enzymatic degradation was observed. Conjugated peptides and peptide/siRNA complexes did not show significant cytotoxicity in selected cell lines. The oleic acid-conjugated peptide showed the highest efficiency in siRNA uptake and silencing of kinesin spindle protein at peptide:siRNA w/w ratio of 80:1 (N/P ≈ 109). The siRNA internalization into non-tumorigenic kidney cells was negligible with all fatty acyl-peptide conjugates. These results indicate that conjugation of fatty acids to CGKRK could create an efficient delivery system for siRNA silencing specifically in tumor cells.

The development of a new class of antibiotics to fight bacterial resistance is a time-consuming effort associated with high-cost and commercial risks. Thus, modification, conjugation or combination of existing antibiotics to enhance their efficacy is a suitable strategy. We have previously reported that the amphiphilic cyclic peptide [R4W4] had antibacterial activity with a minimum inhibitory concentration (MIC) of 2.97 µg/mL against Methicillin-resistant Staphylococcus aureus (MRSA). Herein, we hypothesized that conjugation or combination of the amphiphilic cyclic peptide [R4W4] with levofloxacin or levofloxacin-Q could improve the antibacterial activity of levofloxacin and levofloxacin-Q. Fmoc/tBu solid-phase chemistry was employed to synthesize conjugates of [R4W4K]-levofloxacin-Q and [R4W4K]-levofloxacin. The carboxylic acid group of levofloxacin or levofloxacin-Q was conjugated with the amino group of β-alanine attached to lysine in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) for 3 h to afford the products. Antibacterial assays were conducted to determine the potency of conjugates [R4W4K]-levofloxacin-Q and [R4W4K]-levofloxacin against MRSA and Klebsiella pneumoniae. Although levofloxacin-Q was inactive even at a concentration of 128 µg/mL, [R4W4K]-levofloxacin-Q conjugate and the corresponding physical mixture showed MIC values of 8 µg/mL and 32 µg/mL against MRSA and Klebsiella pneumonia, respectively, possibly due to the activity of the peptide. On the other hand, [R4W4K]-levofloxacin conjugate (MIC = 32 µg/mL and MIC = 128 µg/mL) and the physical mixture (MIC = 8 µg/mL and 32 µg/mL) was less active than levofloxacin (MIC = 2 µg/mL and 4 = µg/mL) against MRSA and Klebsiella pneumoniae, respectively. The data showed that the conjugation of levofloxacin with [R4W4K] significantly reduced the antibacterial activity compared to the parent analogs, while [R4W4K]-levofloxacin-Q conjugate was more significantly potent than levofloxacin-Q alone.

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.

Two bicyclic peptides composed of tryptophan and arginine residues were synthesized from monocyclic peptide building blocks and evaluated as cellular delivery agents. [W5G]-(triazole)-[KR5] and [W5E]-(β-Ala)-[KR5] containing triazole and β-alanine linkers improved the cellular delivery of fluorescein (F')-labeled phosphopeptide F'-GpYEEI (F'-PP) by 7.6- and 19.3-fold, respectively, in human ovarian adenocarcinoma cells. However, parent monocyclic peptide [R5 ] and monocyclic peptide [WR]4 only enhanced the cellular uptake of the phosphopeptide by only 1.3- and 3.7-fold, respectively. Confocal microscopy showed that the corresponding fluorescein-labeled bicyclic peptide F'-[KW4E]-(β-Ala)-[KR5] was localized in the cytosol and nucleus. Studying the cellular uptake of F'-[KW4E]-(β-Ala)-[KR5] in the presence of endocytosis inhibitors indicated that the clathrin- and caveolin-dependent endocytosis are the main pathways for cellular uptake. The bicyclic peptide was able to improve antiproliferative activity of doxorubicin by 20 %. These data suggest that this bicyclic peptide can be utilized as a new class of cell-penetrating peptides and cellular delivery tools.

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.

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 mM in CCRF-CEM cells. These data suggest the potential antiproliferative activity of spiro-pyrrolidines/pyrrolizines.

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 IC 50 value of less than 25 μM. Compound 1-[2-(dimethylamino)ethyl]-5-(2-hydroxy-4-methoxybenzoyl)pyridin-2(1H)-one ( 36 ) exhibited the highest c-Src kinase inhibition with an IC 50 value of 12.5 μM. 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 (300 μM). These results provide insights for further optimization of this scaffold for designing the next generation of 2-pyridone derivatives as candidate Src kinase inhibitors.

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.

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 IC

Gold nanoparticles (AuNPs) were synthesized in situ in a green and rapid method from the reaction of reducing linear and cyclic peptides containing tryptophan and lysine residues, (KW)5 and cyclic [KW]5, with an aqueous solution of HAuCl4 and were evaluated as cellular nanodrug delivery systems. The cyclic or linear nature of the peptide was found to determine the morphology and size of the formed peptide-AuNPs and their in vitro molecular transporting efficiency. While cyclic [KW]5-AuNPs formed sponge-like agglomerates, linear (KW)5-AuNPs demonstrated ball-shaped structures. A comparative flow cytometry study showed that the cellular uptake of fluorescence-labeled anti-HIV drugs (emtricitabine (FTC) and lamivudine (3TC)) in human Leukemia (CCRF-CEM) cells, and a negatively charged cell-impermeable phosphopeptide (GpYEEI) in human ovarian adecarcinoma (SK-OV-3) cells was significantly higher in the presence of cyclic [KW]5-AuNPs than that of linear (KW)5-AuNPs, parent cyclic [KW]5, and linear (KW)5 peptides. For example, the cellular uptake of F′-GpYEEI was enhanced 12.8-fold by c[KW]5-AuNPs. Confocal microscopy revealed the localization of fluorescence-labeled-3TC in the presence of c[KW]5-AuNPs mostly in nucleus in SK-OV-3 cells after 1 h. On the other hand, l(KW)5-AuNPs delivered fluorescence-labeled-3TC in cytoplasm. These data suggest that non-cell penetrating peptides can be converted to efficient molecular transporters through peptide-capped AuNPs formation.

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.

A series of peracetylated O -aryl α,β- d -ribofuranosides have been synthesized and an efficient biocatalytic methodology has been developed for the separation of their anomers which was otherwise almost impossible by column chromatographic or other techniques. The incubation of 2,3,5-tri- O -acetyl-1- O -aryl-α,β- d -ribofuranoside with Lipozyme® TL IM immobilized on silica led to the selective deacetylation of only one acetoxy group, viz the C-5′- O -acetoxy group of the α-anomer over the other acetoxy groups derived from the two secondary hydroxyl groups present in the molecule and also over three acetoxy groups (derived from one primary and two secondary hydroxyls of the β-anomer). This methodology led to the easy synthesis of both, α- and β-anomers of O -aryl d -ribofuranosides. All the arylribofuranosides were screened for inhibition of Src kinase. 1- O -(3-Methoxyphenyl)-β- d -ribofuranoside exhibited the highest activity for inhibition of Src kinase (IC 50 = 95.0 μM).

Derivatives of dasatinib were synthesized via esterification with 25 carboxylic acids including amino acids and fatty acids by extending the inhibitor to interact with more diverse sites and to improve specificity. Dasatinib-L-arginine derivative (Das-R, 7) was the most potent of the inhibitors tested with IC50 values of 4.4 nM

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.

Three fatty acyl conjugates of (-)-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC, emtricitabine) were synthesized and evaluated against HIV-1 cell-free and cell-associated virus and compared with the corresponding parent nucleoside and physical mixtures of FTC and fatty acids. Among all the compounds, the myristoylated conjugate of FTC (5, EC(50) = 0.07-3.7 μM) displayed the highest potency. Compound 5 exhibited 10-24 and 3-13-times higher anti-HIV activity than FTC alone (EC(50) = 0.7-88.6 μM) and the corresponding physical mixtures of FTC and myristic acid (14, EC(50) = 0.2-20 μM), respectively. Cellular uptake studies confirmed that compound 5 accumulated intracellularly after 1 h of incubation and underwent intracellular hydrolysis in CCRF-CEM cells. Alternative studies were conducted using the carboxyfluorescein conjugated with FTC though β-alanine (12) and 12-aminododecanoic acid (13). Acylation of FTC with a long-chain fatty acid in 13 improved its cellular uptake by 8.5-20 fold in comparison to 12 with a short-chain β-alanine. Compound 5 (IC(90) = 15.7-16.1 nM) showed 6.6- and 35.2 times higher activity than FTC (IC(90) = 103-567 nM) against multidrug resistant viruses B-NNRTI and B-K65R, indicating that FTC conjugation with myristic acid generates a more potent analogue with a better resistance profile than its parent compound.

Three nucleoside analogues, 3'-fluoro-2',3'-dideoxythymidine (FLT), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-dideoxy-3'-thiacytidine (3TC) were conjugated with three different dicarboxylic acids to afford the long chain dicarboxylate esters of nucleosides. In general, dinucleoside ester conjugates of FLT and 3TC with long chain dicarboxylic acids exhibited higher anti-HIV activity than their parent nucleosides. Dodecanoate and tetradecanoate dinucleoside ester derivatives of FLT were found to be the most potent compounds with EC(50) values of 0.8-1.0 nM and 3-4 nM against HIV-1(US/92/727) and HIV-1(IIIB) cells, respectively. The anti-HIV activity of the 3TC conjugates containing long chain dicarboxylate diester (EC(50)=3-60 nM) was improved by 1.5-66 fold when compared to 3TC (EC(50)=90-200 nM). This study reveals that the symmetrical ester conjugation of dicarboxylic acids with a number of nucleosides results in conjugates with improved anti-HIV profile.

Protein kinases are enzymes that catalyze the transfer of the γ-phosphate group from ATP to the hydroxyl groups in side chains of tyrosine, serine, or threonine. Protein kinases are divided in two classes: tyrosine kinases (TKs) and serine/threonine kinases (STKs). Overexpression or activation of protein tyrosine kinases (PTKs) has been found to be responsible for the development of many diseases, including cancer, inflammation, and many cardiovascular and neurodegenerative disorders. Thus, the design of PTK inhibitors (PTKIs) has become a subject of a major interest for the pharmaceutical industry. A number of marketed PTKIs that target conserved ATP binding site of PTKs were found to demonstrate toxicity (e.g., imitanib and sorafenib) or to generate resistance (e.g., imitanib and vemurafenib in chronic myeloid leukemia and metastatic melanoma, respectively). Thus, alternative strategies are urgently required for designing novel PTKIs. Linear peptides designed based on the natural protein substrates of PTKs have been introduced to target unique and non conserved PTK regions, such as substrate binding site. These compounds are more specific than the small molecules that usually target conserved ATP binding site. On the other hand, linear peptides are susceptible to hydrolysis by endogenous peptidases. Cyclization of linear peptides has led to generation of diverse conformationally constrained structures as PTKIs. Introduction of the conformational constraints enhances the stability towards proteases, the free energy upon binding, and binding affinity, but reduces the conformational entropy penalty upon receptor binding. Herein, design strategies for conformationally constrained peptides and their application as PTKIs are discussed.

A number of lipophilic 14-substituted derivatives of doxorubicin were synthesized through conjugation of doxorubicin-14-hemisuccinate with different fatty amines or tetradecanol to enhance the lipophilicity, cellular uptake, and cellular retention for sustained anticancer activity. The conjugates inhibited the cell proliferation of human leukemia (CCRF-CEM, 69-76%), colon adenocarcinoma (HT-29, 60-77%), and breast adenocarcinoma (MDA-MB-361, 66-71%) cells at a concentration of 1 μM after 96-120 h of incubation. The N-tetradecylamido derivative of doxorubicin 14-succinate (10) exhibited consistently comparable antiproliferative activity to doxorubicin in a time-dependent manner (IC(50) = 77 nM in CCRF-CEM cells). Flow cytometry analysis showed a 3-fold more cellular uptake of 10 than doxorubicin in SK-OV-3 cells. Confocal microscopy revealed that the conjugate was distributed in cytoplasmic and perinuclear areas during the first 1 h of incubation and slowly relocalized in the nucleus after 24 h. The cellular hydrolysis study showed that 98% of compound 10 was hydrolyzed intracellularly within 48 h and released doxorubicin.

A number of 2-substituted tetrahydroindazolones were synthesized by three-component condensation reaction of 1,3-diketones, substituted hydrazines, benzaldehydes, and Yb(OTf)(3) as a catalyst in [bmim][BF(4)] ionic liquid using a simple, efficient, and economical one-pot method. The synthesized tetrahydroindazolones were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), human ovarian adenocarcinoma (SK-OV-3), and c-Src kinase activity. 3,4-Dichlorophenyl tetrahydroindazolone derivative (15) inhibited the cell proliferation of HT-29 and SK-OV-3 cells by 62% and 58%, respectively. 2,3-Diphenylsubstituted tetrahydroindazolone derivatives, inhibited the cell proliferation of HT-29 cells by 65-72% at a concentration of 50 μM. In general, the tetrahydroindazolones showed modest inhibition of c-Src kinase where 4-tertbutylphenyl- and 3,4-dichlorophenyl- derivatives showed the inhibition of c-Src kinase with IC(50) values of 35.1 and 50.7 μM, respectively.