Publications by authors named "Saghar Mozaffari"

13 Publications

  • Page 1 of 1

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

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

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

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

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

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

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 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

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
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