Publications by authors named "Keykavous Parang"

155 Publications

Design and application of hybrid cyclic-linear peptide-doxorubicin conjugates as a strategy to overcome doxorubicin resistance and toxicity.

Eur J Med Chem 2021 Sep 10;226:113836. Epub 2021 Sep 10.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA, 92618, USA. Electronic address:

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 [RK]WA and linear peptide RKWA were conjugated with Dox through a glutarate linker to afford [RK]WA-Dox and RKWA-Dox conjugates to generate Dox derivatives. Alternatively, [RK]WC was conjugated with Dox via a disulfide linker to generate [RK]WC-S-S-Dox conjugate, where S-S is a disulfide bond. Comparative antiproliferative assays between conjugates [RK]WA-Dox, [RK]WC-S-S-Dox, linear RKWA-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [RK]WA-Dox conjugate was 2-fold more efficient than RKWA-Dox, and [RK]WC-S-S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [RK]WA-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 [RK]WA-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, <9%) at concentrations of 5-10 μM (compared to free Dox at 5 μM that reduced the viability of kidney and heart cells by 85% and 44%, respectively). The fluorescence microscopy images were consistent with the cytotoxicity studies, indicating minimal uptake of the cyclic-linear [RK]WA-Dox (5 μM) in H9C2 cells. In comparison, Dox (5 μM) showed significant uptake, reduced cell viability, and changed the morphology of the cells after 24 h. [RK]WA-Dox showed 16-fold and 9.5-fold higher activity against Dox-resistant cells MDA231R and MES-SA/MX2 (lethal dose for 50% cell death or LC of 2.3 and 4.3 μM, respectively) compared to free Dox (LC of 36-41 μM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [RK]WA-Dox to free Dox in leukemia, ovarian, and gastric cancer cells, significantly reduced toxicity in normal kidney LLC-PK1 and heart H9C2 cells, and significantly higher efficiency in Dox-resistant cells. A number of endocytosis inhibitors did not affect the cellular uptake of [RK]WA-Dox.
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http://dx.doi.org/10.1016/j.ejmech.2021.113836DOI Listing
September 2021

Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positively Charged and Hydrophobic Residues as Molecular Transporters.

Mol Pharm 2021 Sep 7. Epub 2021 Sep 7.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, California 92618, United States.

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)]K(RW) ( = 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)K](RW) (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)K](RW)) 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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.1c00594DOI Listing
September 2021

Exploiting Dengue Virus Protease as a Therapeutic Target; Current Status, Challenges and Future Avenues.

Curr Med Chem 2021 Jun 29. Epub 2021 Jun 29.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan.

Dengue, the oldest and the most prevalent mosquito-borne illness, is caused by the dengue virus (DENV) from the family of Flaviviridae. It infects approximately 400 million individuals per annum, with approximately half of the global population residing in high-risk areas. The factors attributed to the geographic expansion of dengue include urbanization, population density, modern means of transportation, international travels, habit modification, climate change, virus genetics, vector capacity, and poor vector control. Despite the significant progress made in the past against dengue, no effective antiviral therapy is currently available. Among the structural and non-structural proteins encoded by the DENV genome, the NS2B-NS3 protease complex is amongst the extensively studied targets for the development of antiviral therapeutics due to its multiple roles in virus lifecycle. Furthermore, protease inhibitors were found to be successful in Hepatitis C Virus (HCV) and Human Immuno-deficiency Virus (HIV). Likewise, several peptidic, peptide-derived/peptidomimetic, and small molecules inhibitors have been identified as DENV protease inhibitors. Unfortunately, none of them have resulted in a clinically approved drug. Considering all the above-mentioned facts, this review descriptively explains the molecular mechanism and therapeutic potential of DENV protease along with an up to date information on various competitive inhibitors reported against DENV protease. This review might be helpful for the researchers working in this area understand the critical aspects of DENV protease that will help them develop effective and novel inhibitors against DENV to protect the lives of millions of people worldwide.
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http://dx.doi.org/10.2174/0929867328666210629152929DOI Listing
June 2021

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

J Chem Inf Model 2021 06 17;61(6):3015-3026. Epub 2021 May 17.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, California 92618, United States.

Under-expression or overexpression of protein kinases has been shown to be associated with unregulated cell signal transduction in cancer cells. Therefore, there is major interest in designing protein kinase inhibitors as anticancer agents. We have previously reported [WR], 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] ( = 6-9) and hybrid cyclic-linear peptides, [RK]W and [RK]W, containing R and W residues were evaluated for their protein kinase inhibitory potency. Among all the peptides, cyclic peptide [WR] was found to be the most potent tyrosine kinase inhibitor. [WR] showed higher inhibitory activity (IC = 0.21 μM) than [WR], [WR], [WR], and [WR] with IC values of 0.81, 0.57, 0.35, and 0.33 μM, respectively, against c-Src kinase as determined by a radioactive assay using [γ-P]ATP. Consistent with the result above, [WR] inhibited other protein kinases such as Abl kinase activity with an IC value of 0.35 μM, showing 2.2-fold higher inhibition than [WR] (IC = 0.79 μM). [WR] also inhibited PKCa kinase activity with an IC value of 2.86 μM, approximately threefold higher inhibition than [WR] (IC = 8.52 μM). A similar pattern was observed against Braf, c-Src, Cdk2/cyclin A1, and Lck. [WR] exhibited IC values of <0.25 μM against Akt1, Alk, and Btk. These data suggest that [WR] is consistently more potent than other cyclic peptides with a smaller ring size and hybrid cyclic-linear peptides [RK]W and [RK]W against selected protein kinases. Thus, the presence of R and W residues in the ring, ring size, and the number of amino acids in the structure of the cyclic peptide were found to be critical in protein kinase inhibitory potency. We identified three putative binding pockets through automated blind docking of cyclic peptides [WR]. The most populated pocket is located between the SH2, SH3, and N-lobe domains on the opposite side of the ATP binding site. The second putative pocket is formed by the same domains and located on the ATP binding site side of the protein. Finally, a third pocket was identified between the SH2 and SH3 domains. These results are consistent with the non-competitive nature of the inhibition displayed by these molecules. Molecular dynamics simulations of the protein-peptide complexes indicate that the presence of either [WR] or [WR] affects the plasticity of the protein and in particular the volume of the ATP binding site pocket in different ways. These results suggest that the second pocket is most likely the site where these peptides bind and offer a plausible rationale for the increased affinity of [WR].
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http://dx.doi.org/10.1021/acs.jcim.1c00320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238896PMC
June 2021

Cytoplasmic synthesis of endogenous complementary DNA via reverse transcription and implications in age-related macular degeneration.

Proc Natl Acad Sci U S A 2021 02;118(6)

Center for Advanced Vision Science, School of Medicine, University of Virginia, Charlottesville, VA 22908.

retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether cDNA is synthesized independently of genomic integration is unknown. RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed cDNA independently of retrotransposition. RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. reverse transcription can be initiated in the cytoplasm via self-priming of 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 replication cycle shunt as potential therapies for a major cause of blindness.
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http://dx.doi.org/10.1073/pnas.2022751118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017980PMC
February 2021

Peptide/Lipid-Associated Nucleic Acids (PLANAs) as a Multicomponent siRNA Delivery System.

Mol Pharm 2021 03 26;18(3):986-1002. Epub 2021 Jan 26.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, California 92618, United States.

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 naïve 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 .
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00969DOI Listing
March 2021

A Global Review on Short Peptides: Frontiers and Perspectives.

Molecules 2021 Jan 15;26(2). Epub 2021 Jan 15.

School of Chemistry & Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia.

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.
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http://dx.doi.org/10.3390/molecules26020430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830668PMC
January 2021

Click-Free Synthesis of a Multivalent Tricyclic Peptide as a Molecular Transporter.

Pharmaceutics 2020 Sep 3;12(9). Epub 2020 Sep 3.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Harry and Diane Rinker Health Science Campus, Chapman University School of Pharmacy, Irvine, CA 92618, USA.

The cellular delivery of cell-impermeable and water-insoluble molecules remains an ongoing challenge to overcome. Previously, we reported amphipathic cyclic peptides [WR] and [WR] consisting of alternate arginine and tryptophan residues as nuclear-targeting molecular transporters. These peptides contain an optimal balance of positive charge and hydrophobicity, which is required for interactions with the phospholipid bilayer to facilitate their application as a drug delivery system. To further optimize them, we synthesized and evaluated a multivalent tricyclic peptide as an efficient molecular transporter. The monomeric cyclic peptide building blocks were synthesized using Fmoc/tBu solid-phase chemistry and cyclization in the solution and conjugated with each other through an amide bond to afford the tricyclic peptide, which demonstrated modest antibacterial activity against methicillin-resistant (MRSA), , and () with a minimum inhibitory concentration (MIC) of 64-128 µg/mL. The tricyclic peptide was found to be nontoxic up to 30 µM in the breast cancer cell lines (MDA-MB-231). The presence of tricyclic peptide enhanced cellular uptakes of fluorescently-labeled phosphopeptide (F'-GpYEEI, 18-fold), anti-HIV drugs (lamivudine (F'-3TC), emtricitabine (F'-FTC), and stavudine (F'-d4T), 1.7-12-fold), and siRNA (3.3-fold) in the MDA-MB-231 cell lines.
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http://dx.doi.org/10.3390/pharmaceutics12090842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558522PMC
September 2020

Hydrophobic interactions between the HA helix and S4-S5 linker modulate apparent Ca sensitivity of SK2 channels.

Acta Physiol (Oxf) 2021 01 10;231(1):e13552. Epub 2020 Sep 10.

Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, CA, USA.

Aim: Small-conductance Ca -activated potassium (SK) channels are activated exclusively by increases in intracellular Ca that binds to calmodulin constitutively associated with the channel. Wild-type SK2 channels are activated by Ca with an EC value of ~0.3 μmol/L. Here, we investigate hydrophobic interactions between the HA helix and the S4-S5 linker as a major determinant of channel apparent Ca sensitivity.

Methods: Site-directed mutagenesis, electrophysiological recordings and molecular dynamic (MD) simulations were utilized.

Results: Mutations that decrease hydrophobicity at the HA-S4-S5 interface lead to Ca hyposensitivity of SK2 channels. Mutations that increase hydrophobicity result in hypersensitivity to Ca . The Ca hypersensitivity of the V407F mutant relies on the interaction of the cognate phenylalanine with the S4-S5 linker in the SK2 channel. Replacing the S4-S5 linker of the SK2 channel with the S4-S5 linker of the SK4 channel results in loss of the hypersensitivity caused by V407F. This difference between the S4-S5 linkers of SK2 and SK4 channels can be partially attributed to I295 equivalent to a valine in the SK4 channel. A N293A mutation in the S4-S5 linker also increases hydrophobicity at the HA-S4-S5 interface and elevates the channel apparent Ca sensitivity. The double N293A/V407F mutations generate a highly Ca sensitive channel, with an EC of 0.02 μmol/L. The MD simulations of this double-mutant channel revealed a larger channel cytoplasmic gate.

Conclusion: The electrophysiological data and MD simulations collectively suggest a crucial role of the interactions between the HA helix and S4-S5 linker in the apparent Ca sensitivity of SK2 channels.
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http://dx.doi.org/10.1111/apha.13552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736289PMC
January 2021

Cyclic Peptide-Gadolinium Nanoparticles for Enhanced Intracellular Delivery.

Pharmaceutics 2020 Aug 21;12(9). Epub 2020 Aug 21.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA.

A cyclic peptide containing one cysteine and five alternating tryptophan and arginine amino acids [(WR)C] was synthesized using Fmoc/tBu solid-phase methodology. The ability of the synthesized cyclic peptide to produce gadolinium nanoparticles through an in situ one-pot mixing of an aqueous solution of GdCl with [(WR)C] peptide solution was evaluated. Transmission electron microscopy showed the formed peptide-Gd nanoparticles in star-shape morphology with a size of ~250 nm. Flow cytometry investigation showed that the cellular uptake of a cell-impermeable fluorescence-labeled phosphopeptide (F'-GpYEEI, where F' = fluorescein) was approximately six times higher in the presence of [(WR)C]-Gd nanoparticles than those of F'-GpYEEI alone in human leukemia adenocarcinoma (CCRF-CEM) cells after 2 h incubation. The antiproliferative activities of cisplatin and carboplatin (5 µM) were increased in the presence of [(WR)C]-GdNPs (50 μM) by 41% and 18%, respectively, after 72-h incubation in CCRF-CEM cells. The intracellular release of epirubicin, an anticancer drug, from the complex showed that 15% and 60% of the drug was released intracellularly within 12 and 48 h, respectively. This report provides insight about using a non-toxic MRI agent, gadolinium nanoparticles, for the delivery of various types of molecular cargos.
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http://dx.doi.org/10.3390/pharmaceutics12090792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7557599PMC
August 2020

Comparative Molecular Transporter Properties of Cyclic Peptides Containing Tryptophan and Arginine Residues Formed through Disulfide Cyclization.

Molecules 2020 Jun 2;25(11). Epub 2020 Jun 2.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA.

We have previously reported cyclic cell-penetrating peptides [WR] and [WR] 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)C] and linear counterparts (C(WR)C), where x = 4-5, were synthesized using Fmoc/Bu solid-phase peptide synthesis, purified, and characterized. The compounds did not show any significant cytotoxicity (at 25 µM) 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)C] and linear (C(WR)C) demonstrated comparable molecular transporter properties versus [WR] in the delivery of a phosphopeptide (F'-GpYEEI) in CCRF-CEM cells. The uptake of F'-GpYEEI in the presence of 1,4-dithiothreitol (DTT) as the reducing agent was significantly improved in case of l(C(WR)C), while it was not changed by [C(WR)C]. Fluorescence microscopy also demonstrated a significant uptake of F'-GpYEEI in the presence of l(C(WR)C). Cyclic [C(WR)C] improved the uptake of the fluorescent-labeled anti-HIV drugs F'-d4T, F'-3TC, and F'-FTC by 3.0-4.9-fold. These data indicate that both [C(WR)C] and linear (C(WR)C) peptides can act as molecular transporters.
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http://dx.doi.org/10.3390/molecules25112581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321319PMC
June 2020

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

Molecules 2020 May 17;25(10). Epub 2020 May 17.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA.

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'--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 EC value of 0.07 µM against HCoV-229E with TC 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'--fatty acyl conjugates of NRTIs, 5'--tetradecanoyl ester conjugate of FTC showed modest activity with EC and TC values of 72.8 µM and 87.5 µM, respectively. These data can be used for the design of potential compounds against other coronaviruses.
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http://dx.doi.org/10.3390/molecules25102343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7287735PMC
May 2020

Phenylpyrazalopyrimidines as Tyrosine Kinase Inhibitors: Synthesis, Antiproliferative Activity, and Molecular Simulations.

Molecules 2020 May 2;25(9). Epub 2020 May 2.

Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Rhode Island, Kingston, RI 02881, USA.

1-(α,β-Alkene)-substituted phenylpyrazolopyrimidine derivatives with acetyl and functionalized phenyl groups at α- and β-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, IC = 21.7-192.1 µM) 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 () inhibited the cell proliferation of HT-29 and SK-OV-3 by 90% and 79%, respectively, at a concentration of 50 µM after 96 h incubation. The compound showed modest inhibitory activity against c-Src (IC = 60.4 µM), Btk (IC = 90.5 µM), and Lck (IC = 110 µM), 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 . 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.
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http://dx.doi.org/10.3390/molecules25092135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249037PMC
May 2020

Demarcation of Sepsis-Induced Peripheral and Central Acidosis with pH (Low) Insertion Cycle Peptide.

J Nucl Med 2020 09 31;61(9):1361-1368. Epub 2020 Jan 31.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York

Acidosis is a key driver for many diseases, including cancer, sepsis, and stroke. The spatiotemporal dynamics of dysregulated pH across disease remain elusive, and current diagnostic strategies do not provide localization of pH alterations. We sought to explore if PET imaging using hydrophobic cyclic peptides that partition into the cellular membrane at low extracellular pH (denoted as pH [low] insertion cycles, or pHLIC) can permit accurate in vivo visualization of acidosis. Acid-sensitive cyclic peptide c[EWC] pHLIC was conjugated to bifunctional maleimide-NO2A and radiolabeled with Cu (half-life, 12.7 h). C57BL/6J mice were administered lipopolysaccharide (15 mg/kg) or saline (vehicle) and serially imaged with [Cu]Cu-c[EWC] over 24 h. Ex vivo autoradiography was performed on resected brain slices and subsequently stained with cresyl violet to enable high-resolution spatial analysis of tracer accumulation. A non-pH-sensitive cell-penetrating control peptide (c[RWC]) was used to confirm specificity of [Cu]Cu-c[EWC]. CD11b (macrophage/microglia) and TMEM119 (microglia) immunostaining was performed to correlate extent of neuroinflammation with [Cu]Cu-c[EWC] PET signal. [Cu]Cu-c[EWC] radiochemical yield and purity were more than 95% and more than 99%, respectively, with molar activity of more than 0.925 MBq/nmol. Significantly increased [Cu]Cu-c[EWC] uptake was observed in lipopolysaccharide-treated mice (vs. vehicle) within peripheral tissues, including blood, lungs, liver, and small intestines ( < 0.001-0.05). Additionally, there was significantly increased [Cu]Cu-c[EWC] uptake in the brains of lipopolysaccharide-treated animals. Autoradiography confirmed increased uptake in the cerebellum, cortex, hippocampus, striatum, and hypothalamus of lipopolysaccharide-treated mice (vs. vehicle). Immunohistochemical analysis revealed microglial or macrophage infiltration, suggesting activation in brain regions containing increased tracer uptake. [Cu]Cu-c[RWC] demonstrated significantly reduced uptake in the brain and periphery of lipopolysaccharide mice compared with the acid-mediated [Cu]Cu-c[EWC] tracer. Here, we demonstrate that a pH-sensitive PET tracer specifically detects acidosis in regions associated with sepsis-driven proinflammatory responses. This study suggests that [Cu]Cu-pHLIC is a valuable tool to noninvasively assess acidosis associated with both central and peripheral innate immune activation.
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http://dx.doi.org/10.2967/jnumed.119.233072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456172PMC
September 2020

PEGylation and Cell-Penetrating Peptides: Glimpse into the Past and Prospects in the Future.

Curr Top Med Chem 2020 ;20(5):337-348

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, United States.

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.
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http://dx.doi.org/10.2174/1568026620666200128142603DOI Listing
December 2020

Design and Biological Evaluation of Colchicine-CD44-Targeted Peptide Conjugate in an In Vitro Model of Crystal Induced Inflammation.

Molecules 2019 Dec 21;25(1). Epub 2019 Dec 21.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA.

Gout is an inflammatory arthritis due to the joint deposition of monosodium urate (MSU) crystals. Phagocytosis of MSU crystals by tissue macrophages results in the generation of reactive oxygen species (ROS) and production of inflammatory cytokines and chemokines. Colchicine use in gout is limited by severe toxicity. CD44 is a transmembrane glycoprotein that is highly expressed in tissue macrophages and may be involved in gout pathogenesis. The P6 peptide is a 20-amino acid residue peptide that binds to CD44. We hypothesized that the conjugation of colchicine to the P6 peptide would reduce its off-target cytotoxicity while preserving its anti-inflammatory effect. A modified version of P6 peptide and colchicine-P6 peptide conjugate were synthesized using Fmoc/tBu solid-phase and solution-phase chemistry, respectively. A glutaryl amide was used as a linker. The P6 peptide was evaluated for its binding to CD44, association, and internalization by macrophages. Cytotoxic effects of P6 peptide, colchicine, and colchicine-P6 peptide on macrophages were compared and the inhibition of ROS generation and interleukin-8 (IL-8) secretion in MSU-stimulated macrophages treated with P6 peptide, colchicine, or colchicine-P6 peptide was studied. We confirmed that the P6 peptide binds to CD44 and its association and internalization by macrophages were CD44-dependent. Colchicine (1, 10, and 25 μM) demonstrated a significant cytotoxic effect on macrophages while the P6 peptide and colchicine-P6 peptide conjugate (1, 10 and 25 μM) did not alter the viability of the macrophages. The P6 peptide (10 and 25 μM) reduced ROS generation and IL-8 secretion mediated by a reduction in MSU phagocytosis by macrophages. The colchicine-P6 peptide significantly reduced ROS generation and IL-8 secretion compared to the P6 peptide alone at 1 and 10 μM concentrations. Conjugation of colchicine to the P6 peptide reduced the cytotoxic effect of colchicine while preserving its anti-inflammatory activity.
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http://dx.doi.org/10.3390/molecules25010046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982808PMC
December 2019

Comparative Molecular Transporter Efficiency of Cyclic Peptides Containing Tryptophan and Arginine Residues.

ACS Omega 2018 Nov 29;3(11):16281-16291. Epub 2018 Nov 29.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, California 92618, United States.

Cyclic peptides containing tryptophan (W) and arginine (R) residues, [WR], [WR], [WR], [WR], and [WR], were synthesized through Fmoc solid-phase chemistry to compare their molecular transporter efficiency. The in vitro cytotoxicity of the peptides was evaluated using human leukemia carcinoma cell line (CCRF-CEM) and normal kidney cell line (LLC-PK1). [WR], [WR], [WR], and [WR] were not significantly cytotoxic to LLC-PK1cells at a concentration of 10 μM after 3 h incubation. Among all the peptides, [WR] was found to be a more efficient transporter than [WR], [WR], [WR], and [WR] in CCRF-CEM cells for delivery of a cell-impermeable fluorescence-labeled negatively charged phosphopeptide (F'-GpYEEI). [WR] (10 μM) improved the cellular uptake of F'-GpYEEI (2 μM) by 20-fold. The cellular uptake of a fluorescent conjugate of [WR], F'-[WRK], was increased in a concentration- and time-dependent pattern in CCRF-CEM cells. The uptake of F'-[WRK] was slightly reduced in CCRF-CEM cells in the presence of different endocytic inhibitors, such as nystatin, 5-(-ethyl--isopropyl)amiloride, chlorpromazine, chloroquine, and methyl β-cyclodextrin. Furthermore, the uptake of F'-[WRK] was shown to be temperature-dependent and slightly adenosine 5'-triphosphate-dependent. The intracellular/cellular localization (in the nucleus and cytoplasm) of F'-[WRK] was confirmed by fluorescent microscopy in CCRF-CEM cells. These studies suggest that large cyclic peptides containing arginine and tryptophan can be used as a molecular transporter of specific compounds.
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http://dx.doi.org/10.1021/acsomega.8b02589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6643651PMC
November 2018

Cyclic Cell-Penetrating Peptides as Efficient Intracellular Drug Delivery Tools.

Mol Pharm 2019 09 8;16(9):3727-3743. Epub 2019 Aug 8.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences , Chapman University School of Pharmacy , Harry and Diane Rinker Health Science Campus, Irvine , California 92618 , United States.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00633DOI Listing
September 2019

EDB-FN Targeted Peptide-Drug Conjugates for Use against Prostate Cancer.

Int J Mol Sci 2019 Jul 4;20(13). Epub 2019 Jul 4.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, 9401 Jeronimo Road, Irvine, CA 92618, USA.

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] 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 by amide cyclization to obtain [KTVRTSADE] using Fmoc/tBu solid-phase chemistry. Optimized targeting ligand 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 to EDB-FN compared to ligand . Therefore, we hypothesized that the EDB-FN targeted peptides ( and ) 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-drug conjugates. Docetaxel (Doce) and doxorubicin (Dox) were used as anticancer drugs. Dox conjugate 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 was found to be less cytotoxic in all the cell lines as compared to drug alone.
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http://dx.doi.org/10.3390/ijms20133291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651341PMC
July 2019

Amphiphilic Peptides for Efficient siRNA Delivery.

Polymers (Basel) 2019 Apr 17;11(4). Epub 2019 Apr 17.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA.

A number of amphiphilic cyclic peptides-[FR], [WR], and [WK]-containing hydrophobic and positively-charged amino acids were synthesized by Fmoc/tBu solid-phase peptide methods and evaluated for their efficiency in intracellular delivery of siRNA to triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, in the presence and absence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Among the peptides, [WR], which contains alternate tryptophan (W) and arginine (R) residues, was found to be the most efficient in the delivery of siRNA by improving the delivery by more than 3-fold when compared to other synthesized cyclic peptides that were not efficient. The data also showed that co-formulation of [WR] with lipid DOPE significantly enhanced the efficiency of siRNA delivery by up to ~2-fold compared to peptide alone. Based on the data indicating the efficiency of [WR] in siRNA delivery, peptides containing arginine residues on the ring and tryptophan residues on the side chain, [RK]W and [RK]W, were also evaluated, and demonstrated improved delivery of siRNA. The presence of DOPE again enhanced the siRNA delivery in most cases. [WR], [RK]W, and [RK]W did not show any significant toxicity in MDA-MB-231, MDA-MB-468, and AU565 WT cells at N/P ratios of 20:1 or less, in the presence and absence of DOPE. Silencing of kinesin spindle protein (KSP) and Janus kinase 2 (JAK2) was evaluated in MDA-MB-231 cells in the presence of the peptides. The addition of DOPE significantly enhanced the silencing efficiency for all selected peptides. In conclusion, peptides containing tryptophan and arginine residues were found to enhance siRNA delivery and to generate silencing of targeted proteins in the presence of DOPE.
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http://dx.doi.org/10.3390/polym11040703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523661PMC
April 2019

Synthesis and Antiproliferative Activities of Conjugates of Paclitaxel and Camptothecin with a Cyclic Cell-Penetrating Peptide.

Molecules 2019 Apr 11;24(7). Epub 2019 Apr 11.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA.

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' hydroxyl group of PTX with glutaric anhydride and conjugated with the cyclic peptide [W(WR)₄K(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 µM concentration after 72 h incubation.
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http://dx.doi.org/10.3390/molecules24071427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480016PMC
April 2019

Antibiotics-Peptide Conjugates Against Multidrug-resistant Bacterial Pathogens.

Curr Top Med Chem 2018 ;18(22):1926-1936

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, 9401 Jeronimo Road, Irvine, CA 92618-1908, United States.

The menace of multi-drug resistance by bacterial pathogens that are responsible for infectious diseases in humans and animals cannot be over-emphasized. Many bacteria develop resistance to antibiotics by one or more combination of resistance mechanisms namely, efflux pump activation thereby reducing bacteria intracellular antibiotic concentration, synthesizing a protein that protects target site causing poor antibiotic affinity to the binding site, or mutations in DNA and topoisomerase gene coding that alters residues in the binding sites. The ability to use a combination of these resistance mechanisms among others creates a phenomenon known as antimicrobial drug resistance. The development of a new class of antibiotics to address bacterial resistance will require many resources, such as time-consuming effort and high cost associated with commercial risk. Hence, the researchers have adopted a strategic approach to enhance the antibacterial efficacy of existing antibiotics by conjugation or combination of existing antibiotics. A number of peptides have become known as antibacterial, cell-penetrating, or membrane-active agents. Antibiotics-Peptide Conjugates (APCs) are a combination of known antibiotics with a peptide connected through a linker. The rationale is to produce an alternative multifunctional antimicrobial compound that will elicit synergistic antibacterial activities while reducing known shortcomings of antibiotics or peptides, such as cellular penetration, serum instability, cytotoxicity, hemolysis, and instability in high salt conditions. In this review, we overview APCs which are used, as a strategy to combat the menace of multi-drug resistance of bacterial pathogens. Furthermore, we explain the focus area of adopted APC strategies and physicochemical properties data that show how they can be used to improve antibacterial efficacy.
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http://dx.doi.org/10.2174/1568026619666181129141524DOI Listing
January 2019

Synthesis and antiproliferative activities of doxorubicin thiol conjugates and doxorubicin-SS-cyclic peptide.

Eur J Med Chem 2019 Jan 23;161:594-606. Epub 2018 Oct 23.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA, 92618, United States. Electronic address:

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)K], were synthesized. Dox was reacted with Traut's 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)K] was prepared using Fmoc solid-phase strategy. Dox-SS-Py was reacted with the free sulfhydryl of cysteine in [C(WR)K] to generate Dox-SS-[C(WR)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)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)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)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)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)K] was reduced in comparison of Dox when co-treated with FeCl. These data indicate that Dox-SH, Dox-SS-Pyr, and Dox-SS-[C(WR)K] have the potential to be further examined as Dox alternatives and anticancer agents.
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http://dx.doi.org/10.1016/j.ejmech.2018.10.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8418874PMC
January 2019

Design, Synthesis, and Evaluation of Amphiphilic Cyclic and Linear Peptides Composed of Hydrophobic and Positively-Charged Amino Acids as Antibacterial Agents.

Molecules 2018 Oct 22;23(10). Epub 2018 Oct 22.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA.

Antimicrobial peptides (AMPs) contain amphipathic structures and are derived from natural resources. AMPs have been found to be effective in treating the infections caused by antibiotic-resistant bacteria (ARB), and thus, are potential lead compounds against ARB. AMPs' physicochemical properties, such as cationic nature, amphiphilicity, and their size, will provide the opportunity to interact with membrane bilayers leading to damage and death of microorganisms. Herein, AMP analogs of [R₄W₄] were designed and synthesized by changing the hydrophobicity and cationic nature of the lead compound with other amino acids to provide insights into a structure-activity relationship against selected model Gram-negative and Gram-positive pathogens. Clinical resistant strains of methicillin-resistant (MRSA) and () were used in the studies. Our results provided information about the structural requirements for optimal activity of the [R₄W₄] template. When tryptophan was replaced with other hydrophobic amino acids, such as phenylalanine, tyrosine, alanine, leucine, and isoleucine, the antibacterial activities were significantly reduced with MIC values of >128 µg/mL. Furthermore, a change in stereochemistry caused by d-arginine, and use of -methyltryptophan, resulted in a two-fold reduction of antibacterial activity. It was found that the presence of tryptophan is critical for antibacterial activity, and could not be substituted with other hydrophobic residues. The study also confirmed that cyclic peptides generally showed higher antibacterial activities when compared with the corresponding linear counterparts. Furthermore, by changing tryptophan numbers in the compound while maintaining a constant number of arginine, we determined the optimal number of tryptophan residues to be four, as shown when the number of tryptophan residues increased, a decrease in activity was observed.
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http://dx.doi.org/10.3390/molecules23102722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222377PMC
October 2018

Difatty Acyl-Conjugated Linear and Cyclic Peptides for siRNA Delivery.

ACS Omega 2017 Oct 19;2(10):6939-6957. Epub 2017 Oct 19.

Department of Biomedical and Pharmaceutical Sciences, Center For Targeted Drug Delivery, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, California 92618, United States.

A number of amphiphilic difatty acyl linear and cyclic RK peptide conjugates were synthesized by solid-phase peptide methods to enhance the interaction with the hydrophobic cellular phospholipid bilayer and to improve siRNA delivery and silencing. Binding to siRNA molecules was significantly less for the cyclic peptide conjugates. A gradual decrease was observed in the particle size of the complexes with increasing peptide/siRNA ratio for most of the synthesized peptides, suggesting the complex formation. Most of the complexes showed a particle size of less than 200 nm, which is considered an appropriate size for in vitro siRNA delivery. A number of fatty acyl-conjugated peptides, such as LP-C16 and LP-C18, displayed near complete protection against serum degradation. Flow cytometry studies demonstrated significantly higher internalization of fluorescence-labeled siRNA (FAM-siRNA) in the presence of LP-C16, LP-C18, and CP-C16 with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) addition. Confocal microscopy confirmed the cellular internalization of fluorescence-labeled siRNA in the presence of LP-C16 and LP-C18 with DOPE when compared with cells exposed to DOPE/FAM-siRNA. While C16- and C18-conjugated peptides (especially linear peptides) showed silencing against kinesin spindle protein (KSP) and janus kinase 2 (JAK2) proteins, the addition of DOPE enhanced the silencing efficiency significantly for all selected peptides, except for CP-C16. In conclusion, C16 and C18 difatty acyl peptide conjugates were found to enhance siRNA delivery and generate silencing of targeted proteins in the presence of DOPE. This study provides insights for the design and potential application of optimized difatty acyl peptide/lipid nanoparticles for effective siRNA delivery.
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http://dx.doi.org/10.1021/acsomega.7b00741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044792PMC
October 2017

Design, Synthesis, and Evaluation of Homochiral Peptides Containing Arginine and Histidine as Molecular Transporters.

Molecules 2018 06 29;23(7). Epub 2018 Jun 29.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA.

Linear (HR) and cyclic [HR] peptides (n = 4,5) containing alternate arginine and histidine residues were synthesized. The peptides showed 0⁻15% cytotoxicity at 5⁻100 µM in human ovarian adenocarcinoma (SK-OV-3) cells while they exhibited 0⁻12% 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 -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/Bu 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, C, C, and C alkyl chain did not show cytotoxicity on SK-OV-3 or CCRF-CEM cell lines up to 50 µM concentration; however, at higher concentration (100 µM), they showed mild cytotoxicity. For example, C-(HR)₄ was also found to reduce the proliferation of SK-OV-3 cells by 11% at 50 µM and C-(HR)₄ showed mild toxicity at 10 µM, reducing the proliferation of SK-OV-3 cells by 21%. Increase in the length of alkyl chain showed cytotoxicity to the cell lines. C-(HR)₄ peptide showed better efficiency in translocation of F′-GpYEEI to SK-OV-3 than the phosphopeptide alone. Further investigation of C-(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.
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http://dx.doi.org/10.3390/molecules23071590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100079PMC
June 2018

Efficient Intracellular Delivery of Cell-Impermeable Cargo Molecules by Peptides Containing Tryptophan and Histidine.

Molecules 2018 06 26;23(7). Epub 2018 Jun 26.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, CA 92618, USA.

We have previously evaluated and reported numerous classes of linear and cyclic peptides containing hydrophobic and hydrophilic segments for intracellular delivery of multiple molecular cargos. Herein, a combination of histidine and tryptophan amino acids were designed and evaluated for their efficiency in intracellular delivery of cell-impermeable phosphopeptides and the anti-HIV drug, emtricitabine. Two new decapeptides, with linear and cyclic natures, both containing alternate tryptophan and histidine residues, were synthesized using Fmoc/tBu solid-phase chemistry. The peptides were characterized and purified by using matrix-assisted laser desorption/ionization (MALDI) spectroscopy and high-performance liquid chromatography (HPLC), respectively. These peptides did not show significant toxicity up to 100 µM in ovarian cancer (SK-OV-3) and leukemia cancer (CCRF-CEM) cells. Furthermore, the cellular uptake of a fluorescence (F’)-labeled cell-impermeable phosphopeptide (F’-GpYEEI) was enhanced in the presence of linear (WH)₅ and cyclic [WH]₅ by 2- and 8-fold, respectively, compared to the uptake of the phosphopeptide alone. The cellular uptake was not significantly changed in the presence of endocytosis inhibitors. Furthermore, the intracellular uptake of the fluorescently-labeled anti-HIV drug, emtricitabine (F’-FTC), by linear (WH)₅ and cyclic [WH]₅ in SK-OV-3 cancer cell lines was found to be enhanced by 3.5- and 9-fold, respectively, compared to that of the drug alone. Fluorescent uptake experiments confirmed the localization of F’-GpYEEI-loaded cyclic [WH]₅ intracellularly in the SK-OV-3 cancer cell line after 3 h of incubation. Thus, these data demonstrated that [WH]₅ containing tryptophan and histidine enhanced the cellular uptake of F’-GpYEEI and emtricitabine.
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http://dx.doi.org/10.3390/molecules23071536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100250PMC
June 2018

TrkB-enhancer facilitates functional recovery after traumatic brain injury.

Sci Rep 2017 09 8;7(1):10995. Epub 2017 Sep 8.

Neurotrauma and Brain Barriers Research Laboratory, Department of Emergency Medicine, Alpert Medical School of Brown University, Providence, RI, 02903, USA.

Brain-derived neurotrophic factor (BDNF), a key player in regulating synaptic strength and learning, is dysregulated following traumatic brain injury (TBI), suggesting that stimulation of BDNF signaling pathways may facilitate functional recovery. This study investigates whether CN2097, a peptidomimetic ligand which targets the synaptic scaffold protein, postsynaptic density protein 95, to enhance downstream signaling of tropomyosin-related kinase B, a receptor for BDNF, can improve neurological function after TBI. Moderate to severe TBI elicits neuroinflammation and c-Jun-N-terminal kinase (JNK) activation, which is associated with memory deficits. Here we demonstrate that CN2097 significantly reduces the post-traumatic synthesis of proinflammatory mediators and inhibits the post-traumatic activation of JNK in a rodent model of TBI. The recordings of field excitatory post-synaptic potentials in the hippocampal CA1 subfield demonstrate that TBI inhibits the expression of long-term potentiation (LTP) evoked by high-frequency stimulation of Schaffer collaterals, and that CN2097 attenuates this LTP impairment. Lastly, we demonstrate that CN2097 significantly improves the complex auditory processing deficits, which are impaired after injury. The multifunctionality of CN2097 strongly suggests that CN2097 could be highly efficacious in targeting complex secondary injury processes resulting from neurotrauma.
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http://dx.doi.org/10.1038/s41598-017-11316-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591207PMC
September 2017

Efficient synthesis of CN2097 using in situ activation of sulfhydryl group.

Tetrahedron Lett 2017 08 23;58(31):3053-3056. Epub 2017 Jun 23.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, California 92618, USA.

CN2097 (RCs-sCYK[KTE(β-Ala)]V) is a rationally designed peptidomimetic that shows effectiveness in preclinical models for the treatment of neurological disorders, such as Angelman syndrome, traumatic brain injury (TBI) and stroke. Because of its therapeutic activity for the treatment of human CNS disorders, there was an urgent need to develop an efficient strategy for large-scale synthesis of CN2097. The synthesis of CN2097 was accomplished using Fmoc/tBu solid phase chemistry in multiple steps. Two different peptide fragments (activated polyarginine peptide Npys-sCR and CYK[KTE(β-Ala)]V) were synthesized, followed by solution phase coupling in water. Activation of the polyarginine (CR) was achieved in situ during cleavage of protected peptide (C(Trt)R(Pbf)) from the Rink amide resin using 5 equiv. of 2,2-dithopyridine in TFA:TIS:HO (95:2.5:2.5, v/v/v) for 4 h. The disulfide coupling was efficient which provided a 60% yield.
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http://dx.doi.org/10.1016/j.tetlet.2017.06.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557301PMC
August 2017

Tumor-targeted delivery of siRNA using fatty acyl-CGKRK peptide conjugates.

Sci Rep 2017 07 21;7(1):6093. Epub 2017 Jul 21.

Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, California, 92618, United States.

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.
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http://dx.doi.org/10.1038/s41598-017-06381-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522445PMC
July 2017
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