Targeting a c-Myc inhibitory polypeptide to specific intracellular compartments using cell penetrating peptides.
ABSTRACT The therapeutic index of current anti-cancer chemotherapeutics can be improved by two major mechanisms: 1) developing drugs which are specifically toxic to the cancer cells and 2) developing methods to deliver drugs to the tumor site. In an attempt to combine these approaches, we developed a thermally responsive polypeptide inhibitor of c-Myc. This polypeptide is based on the thermally responsive Elastin-like polypeptide (ELP). When injected systemically, ELP-fused drugs will aggregate and accumulate at the tumor site where local hyperthermia is applied. ELP was fused to a peptide which blocks c-Myc/Max dimerization (H1), thereby inhibiting transcription activation by c-Myc (ELP-H1). In this study, the cellular uptake, intracellular distribution, and potency of the Pen, Tat and Bac cell penetrating peptides fused to ELP-H1 were evaluated. While Pen-ELP-H1 and Tat-ELP-H1 were localized in the cytoplasm, Bac-ELP-H1 localized to the nucleus in a subset of the cells and was the most potent inhibitor of MCF-7 cell proliferation. This data demonstrates that ELP can be targeted to the desired cellular compartment simply by choice of the CPP used, resulting in a more potent nuclear targeted c-Myc inhibitory polypeptide which may be beneficial in cancer therapy.
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ABSTRACT: The last several decades have seen intensive research into the molecular mechanisms underlying the symptoms of preeclampsia. While the underlying cause of preeclampsia is believed to be defective placental development and resulting placental ischemia, it is only recently that the links between the ischemic placenta and maternal symptomatic manifestation have been elucidated. Several different pathways have been implicated in the development of the disorder; most notably production of the anti-angiogenic protein sFlt-1, induction of auto-immunity and inflammation, and production of reactive oxygen species. While the molecular mechanisms are becoming clearer, translating that knowledge into effective therapeutics has proven elusive. Here we describe a number of peptide based therapies we have developed to target theses pathways, and which are currently being tested in preclinical models. These therapeutics are based on a synthetic polymeric carrier elastin-like polypeptide (ELP), which can be synthesized in various sequences and sizes to stabilize the therapeutic peptide and avoid crossing the placental interface. This prevents fetal exposure and potential developmental effects. The therapeutics designed will target known pathogenic pathways, and the ELP carrier could prove to be a versatile delivery system for administration of a variety of therapeutics during pregnancy.Frontiers in Pharmacology 09/2014; 5:201. DOI:10.3389/fphar.2014.00201
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ABSTRACT: Abstract Background: Pregnant females are largely overlooked in drug development due to concerns for fetal health. Additionally, pregnancy is often an exclusion criterion in clinical trials, so the safety of many drugs during pregnancy is unknown. Purpose: The goal of this study was to evaluate Elastin-like Polypeptide (ELP), a synthetic protein derived from human elastin, for maternally sequestered drug delivery. ELP is a versatile drug carrier with a long plasma half-life, low immunogenicity, and the ability to be fused to nearly any small molecule or protein-based therapeutic. Methods: We determined the pharmacokinetics, biodistribution, and fetal exposure to the ELP drug carrier using quantitative fluorescence techniques in a rat pregnancy model. Results: After either bolus IV administration or continuous infusion over five days, ELPs accumulated strongly in the kidneys, liver, and placenta, but importantly, little to no ELPs were detectable in the fetus. Within the placenta, ELPs were localized to the chorionic plate and broadly distributed within the labyrinth, but were excluded from the fetal portion of the chorionic villi. Conclusion: These data indicate that ELP does not cross the placenta, and they suggest that this adaptable drug delivery system is a promising platform for prevention of fetal drug exposure.Journal of Drug Targeting 08/2014; 22(10):1-13. DOI:10.3109/1061186X.2014.950666 · 2.72 Impact Factor
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ABSTRACT: S100B, a glial-secreted protein, is believed to play a major role in neurodegeneration in Alzheimer's disease, Down syndrome, traumatic brain injury, and spinocerebellar ataxia type 1 (SCA1). SCA1 is a trinucleotide repeat disorder in which the expanded polyglutamine mutation in the protein ataxin-1 primarily targets Purkinje cells of the cerebellum. Currently, the exact mechanism of S100B-mediated Purkinje cell damage in SCA1 is not clear. However, here we show that S100B may act via the activation of the receptor for advanced glycation end product (RAGE) signaling pathway, resulting in oxidative stress-mediated injury to mutant ataxin-1-expressing neurons. To combat S100B-mediated neurodegeneration, we have designed a selective thermally responsive S100B inhibitory peptide, Synb1-ELP-TRTK. Our therapeutic polypeptide was developed using three key elements: (1) the elastin-like polypeptide (ELP), a thermally responsive polypeptide, (2) the TRTK12 peptide, a known S100B inhibitory peptide, and (3) a cell-penetrating peptide, Synb1, to enhance intracellular delivery. Binding studies revealed that our peptide, Synb1-ELP-TRTK, interacts with its molecular target S100B and maintains a high S100B binding affinity as comparable with the TRTK12 peptide alone. In addition, in vitro studies revealed that Synb1-ELP-TRTK treatment reduces S100B uptake in SHSY5Y cells. Furthermore, the Synb1-ELP-TRTK peptide decreased S100B-induced oxidative damage to mutant ataxin-1-expressing neurons. To test the delivery capabilities of ELP-based therapeutic peptides to the cerebellum, we treated mice with fluorescently labeled Synb1-ELP and observed that thermal targeting enhanced peptide delivery to the cerebellum. Here, we have laid the framework for thermal-based therapeutic targeting to regions of the brain, particularly the cerebellum. Overall, our data suggest that thermal targeting of ELP-based therapeutic peptides to the cerebellum is a novel treatment strategy for cerebellar neurodegenerative disorders.Neuroscience 09/2011; 197:369-80. DOI:10.1016/j.neuroscience.2011.09.025 · 3.33 Impact Factor