Cell-permeable Peptide Research Articles

Autophagy facilitates the release of immunogenic signals following chemotherapy in 3D models of mesothelioma.

We have previously shown that nearly half of mesothelioma patients have tumors with low autophagy and that these patients have a significantly worse outcome than those with high autophagy. We hypothesized that autophagy may be beneficial by facilitating immunogenic cell death (ICD) of tumor cells following chemotherapy. An important hallmark of ICD is that death of tumor cells is preceded or accompanied by the release of damage-associated molecular pattern molecules (DAMPs), which then can stimulate an antitumor immune response. Therefore, we measured how autophagy affected the release of three major DAMPs: high mobility group box 1 (HMGB1), ATP, and calreticulin following chemotherapy. We found that autophagy in three-dimensional (3D) models with low autophagy at baseline could be upregulated with the cell-permeant Tat-BECN1 peptide and confirmed that autophagy in 3D models with high autophagy at baseline could be inhibited with MRT 68921 or ATG7 RNAi, as we have previously shown. In in vitro 3D spheroids, we found that, when autophagy was high or upregulated, DAMPs were released following chemotherapy; however, when autophagy was low or inhibited, DAMPs release was significantly impaired. Similarly, in ex vivo tumors, when autophagy was high or upregulated, HMGB1 was released following chemotherapy but, when autophagy was low, HMGB1 release was not seen. We conclude that autophagy can be upregulated in at least some tumors with low autophagy and that upregulation of autophagy can restore the release of DAMPs following chemotherapy. Autophagy may be necessary for ICD in this tumor.

1728-P: Inhibition of PKC-Theta in Prediabetic NOD Mice Prevents Diabetes Onset and Increases Regulatory CD4 T Cells in a Subset of Animals

The novel PKC isoform, PKC-theta, is particularly enriched in T lymphocyte populations, serving a critical role in signal transduction after CD3/CD28 T cell receptor stimulation. PKC-theta is known to be involved in promoting T cell mediated immunity and has been implicated in other autoimmune diseases. We hypothesized that inhibition of this isoform would increase regulatory T cell phenotypes, promote an anti-inflammatory environment, and lead to delayed onset of type 1 diabetes in the NOD mouse model. A cell permeable pseudosubstrate peptide inhibitor was identified with potent suppression of IL-2 secretion at concentrations ≤15 μg in T cell enriched populations stimulated in vitro with CD3 and CD28 antibodies and a significant reduction in stimulated IL-2 secretion after 7 days of intraperitoneal treatment with 50 μg of pseudosubstrate inhibitor. Five-week-old prediabetic NOD mice were either treated daily with 50 μg of inhibitor or mock treated with saline for 4 weeks, then allowed to progress until 17 weeks of age. An 80% conversion to diabetes, based on IP glucose tolerance tests, was observed in saline mice compared to 30% conversion in inhibitor-treated mice, which correlated with a greater frequency of inflammation-free islets and islets with periinsulitis only in inhibitor-treated mice. Inhibitor-treated mice had increased frequencies of IL-10 secreting cells by ELISPOT, and increased Tregs in spleen at 17 weeks of age while reduced frequencies of IL-17+ CD4+ T cells. This study was repeated with a scrambled peptide control and later endpoint to determine if T1D would be prevented entirely. At 25 weeks of age a 66% conversion to diabetes was observed in scrambled peptide treated mice, while only 33% converted in the peptide treated. Our data demonstrate that PKC theta activation contributes to the progression of beta cell destruction in NOD mice, where inhibition of this isoform may confer an increase in protective regulatory T cell populations. Disclosure J.E. DiLisio: None. N. Mishkin: None. B. Podell: None. Funding National Institutes of Health (5K01OD016997 to B.P.); National Center for Advancing Translational Sciences (UL1TR001082 to B.P.)

Mechanistic study of the superior anti-cancer properties of a first-in-class small molecule targeting PCNA.

e14636 Background: Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was predominantly expressed in a broad range of cancer cells and tumor tissues, but not significantly in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interdomain connector loop of PCNA that is known to be a major binding site for many of PCNA’s interacting proteins. A cell permeable peptide harboring the L126-Y133 sequence inhibited PCNA function in cancer cells and selectively kills cancer cells and xenograft tumors. Methods: Based on these observations, we sought small molecules targeting this peptide region of PCNA as potential broad-spectrum anti-cancer agents. Results: Our effort led to a drug candidate, AOH1996, which selectively kills a broad range of cancer cells at high nanomolar concentrations, but is not associated with significant toxicity to non-malignant cells. It also works synergistically with DNA damaging chemotherapeutic drugs, such as cisplatin and irinotecan, to selectively kill cancer cells. This compound is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it doesn’t cause significant toxicity at 2.5 times its effective dose. Mechanistically, AOH1996 competes with T3, a known PCNA ligand, for binding to PCNA. However, the mechanism by which AOH1996 exerts its effect on cancer cells may not be identical to what have been reported for the T3 analogs. In particular, we found that AOH1996 interferes with the association of PCNA and MCM7 to euchromatin, leading to DNA replication stress, blockade of homologous recombination-mediated DNA repair, and induction of apoptosis in cancer cells. Conclusions: These findings demonstrated the potential of this compound as a novel therapeutic agent warranting clinical investigation for cancer treatment. We have started planning a phase 1 clinical study for this compound.

The selective disruption of presynaptic JNK2/STX1a interaction reduces NMDA receptor-dependent glutamate release

The neuronal loss caused by excessive glutamate release, or ‘excitotoxicity’, leads to several pathological conditions, including cerebral ischemia, epilepsy, and neurodegenerative diseases. Over-stimulation of presynaptic N-methyl-D-aspartate (NMDA) receptors is known to trigger and support glutamate spillover, while postsynaptic NMDA receptors are responsible for the subsequent apoptotic cascade. Almost all molecules developed so far are unable to selectively block presynaptic or postsynaptic NMDA receptors, therefore a deeper knowledge about intracellular NMDA pathways is required to design more specific inhibitors. Our previous work showed that presynaptic c-Jun N-terminal kinase 2 (JNK2) specifically regulates NMDA-evoked glutamate release and here we demonstrate that an interaction between Syntaxin-1a and JNK2 is fundamental to this mechanism. Based on this evidence, a new cell permeable peptide (CPP), “JGRi1”, has been developed to disrupt the JNK2/STX1a interaction to indirectly, but specifically, inhibit presynaptic NMDA receptor signaling. JGRi1 reduces the NMDA-evoked release of glutamate both in in-vitro and ex-vivo experiments while also being able to widely diffuse throughout brain tissue via intraperitoneal administration. In conclusion, the JNK2/STX1 interaction is involved in presynaptic NMDA-evoked glutamate release and the novel CPP, JGRi1, acts as a pharmacological tool that promotes neuroprotection.

Specific targeting of PKC\u03b4 suppresses osteoclast differentiation by accelerating proteolysis of membrane-bound macrophage colony-stimulating factor receptor

c-Fms is the macrophage colony-stimulating factor (M-CSF) receptor, and intracellular signalling via the M-CSF/c-Fms axis mediates both innate immunity and bone remodelling. M-CSF-induced transient proteolytic degradation of c-Fms modulates various biological functions, and protein kinase C (PKC) signalling is activated during this proteolytic process via an unknown mechanism. Notably, the role of specific PKC isoforms involved in c-Fms degradation during osteoclast differentiation is not known. Here, we observed that inactivation of PKCδ by the biochemical inhibitor rottlerin, a cell permeable peptide inhibitor, and short hairpin (sh) RNA suppresses osteoclast differentiation triggered by treatment with M-CSF and receptor activator of NF-κB ligand. Interestingly, inhibition of PKCδ by either inhibitor or gene silencing of PKCδ accelerated M-CSF-induced proteolytic degradation of membrane-bound c-Fms via both the lysosomal pathway and regulated intramembrane proteolysis (RIPping), but did not affect c-fms expression at the mRNA level. Degradation of c-Fms induced by PKCδ inactivation subsequently inhibited M-CSF-induced osteoclastogenic signals, such as extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), p38, and Akt. Furthermore, mice administered PKCδ inhibitors into the calvaria periosteum exhibited a decrease in both osteoclast formation on the calvarial bone surface and the calvarial bone marrow cavity, which reflects osteoclastic bone resorption activity. These data suggest that M-CSF-induced PKCδ activation maintains membrane-anchored c-Fms and allows the sequential cellular events of osteoclastogenic signalling, osteoclast formation, and osteoclastic bone resorption.

The cytoplasmic domain of CTLA-4 control autoimmunity via inducing regulatory T cells

Abstract Regulatory T cells (Tregs) have critical roles for maintaining immune tolerance and homeostasis. Recently, it is revealed that CTLA-4 is an essential membrane protein for the suppressive function of Treg. However, the molecular mechanism of cytoplasmic domain of CTLA-4 (ctCTLA-4) and its role in Treg function and differentiation is not clearly understood. In this study, we utilized ctCTLA-4 peptide and its various mutant forms conjugated with dNP2, a cell-permeable peptide, to investigate the mechanism of ctCTLA-4 in Treg function and differentiation. We found that the ctCTLA-4 convert naïve T cells to Foxp3-positive cells in T helper 17 cell differentiation condition in vitro, while the lysine-rich motif mutant form could not. The dNP2-ctCTLA-4 also significantly inhibits experimental autoimmune encephalomyelitis (EAE) in vivo, by reducing CNS-infiltrating Th17 cells with significantly increased Treg proportion. As similar with in vitroresults, lysine-rich motif mutant form could not increase Treg proportion and inhibit EAE. To elucidate its molecular mechanisms, we conducted HuProt protein microarray to identify lysine-rich motif dependent binding partner of ctCTLA-4 related with increasing Tregs. With the results, we found that ctCTLA-4 decreased a phosphorylation of MAPK in T cell stimulation conditions resulting enhanced nuclear localization of Smad2/3, a key transcription factor for Foxp3 expression. These results suggest that ctCTLA-4 has a critical molecular mechanism of inducing Tregs to maintain peripheral immune tolerance to control autoimmunity.

Development of an ORF45-Derived Peptide To Inhibit the Sustained RSK Activation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus.

The lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) requires sustained extracellular signal-regulated kinase (ERK)-p90 ribosomal S6 kinase (RSK) activation, which is induced by an immediate early (IE) gene-encoded tegument protein called ORF45, to promote the late transcription and translation of viral lytic genes. An ORF45-null or single-point F66A mutation in ORF45 abolishes ORF45-RSK interaction and sustained ERK-RSK activation during lytic reactivation and subsequently results in a significant decrease in late lytic gene expression and virion production, indicating that ORF45-mediated RSK activation plays a critical role in KSHV lytic replication. Here, we demonstrate that a short ORF45-derived peptide in the RSK-binding region is sufficient for disrupting ORF45-RSK interaction, consequently suppressing lytic gene expression and virion production. We designed a nontoxic cell-permeable peptide derived from ORF45, TAT-10F10, which is composed of the ORF45 56 to 76 amino acid (aa) region and the HIV Tat protein transduction domain, and this peptide markedly inhibits KSHV lytic replication in iSLK.219 and BCBL1 cells. Importantly, this peptide enhances the inhibitory effect of rapamycin on KSHV-infected cells and decreases spontaneous and hypoxia-induced lytic replication in KSHV-positive lymphoma cells. These findings suggest that a small peptide that disrupts ORF45-RSK interaction might be a promising agent for controlling KSHV lytic infection and pathogenesis.IMPORTANCE ORF45-induced RSK activation plays an essential role in KSHV lytic replication, and ORF45-null or ORF45 F66A mutagenesis that abolishes sustained RSK activation and RSK inhibitors significantly decreases lytic replication, indicating that the ORF45-RSK association is a unique target for KSHV-related diseases. However, the side effects, low affinity, and poor efficacy of RSK modulators limit their clinical application. In this study, we developed a nontoxic cell-permeable ORF45-derived peptide from the RSK-binding region to disrupt ORF45-RSK associations and block ORF45-induced RSK activation without interfering with S6K1 activation. This peptide effectively suppresses spontaneous, hypoxia-induced, or chemically induced KSHV lytic replication and enhances the inhibitory effect of rapamycin on lytic replication and sensitivity to rapamycin in lytic KSHV-infected cells. Our results reveal that the ORF45-RSK signaling axis and KSHV lytic replication can be effectively targeted by a short peptide and provide a specific approach for treating KSHV lytic and persistent infection.

NEMO-binding domain peptides alleviate perihematomal inflammation injury after experimental intracerebral hemorrhage

Inflammation aggravates the lethal consequences of intracerebral hemorrhage. Recently, many studies have found that nuclear factor-κB (NF-κB) is a crucial transcription factor that initiates inflammation in the perihematomal region of ICH. NF-κB essential modulator (NEMO)-binding domain (NBD) peptide, a cell-permeable peptide spanning the NBD of IKKα or IKKβ, functions as a highly specific inhibitor of NF-κB. This peptide can negatively regulate the NF-κB pathway. The present study aimed to explore the effects and underlying pathomechanisms of NBD peptides after ICH. Striatum infusion of whole blood or saline was performed on C57BL/6 mice (n = 198). Experimental animals were administered NBD or control (mutated) peptides 2 h before or after ICH by intracerebroventricular injection (icv.). NBD peptides significantly inhibited edema formation, ameliorated the neurological deficits, markedly reduced IκBα and p65 phosphorylation, blocked nuclear translocation of p65, and upregulated IκBα expression by NF-κB after ICH induction. Using an in vitro hemin toxicity model, we investigated the effects of NBD peptides on microglial inflammation. We found that NBD peptides suppressed microglia inflammation and lowered the expression of TNF-α and IL-1β in both in vivo and in vitro experiments. Further experiments were performed in mice and cultured microglia, which treated with NBD peptides in the presence of p65 siRNA confirmed that the specificity of NBD peptides inhibit ICH-induced NF-κB activation. This study demonstrated that NBD peptides exert a neuroprotective role after ICH and might be a potential candidate for a novel therapeutic strategy for ICH.

Inhibition of the Interaction Between Group I Metabotropic Glutamate Receptors and PDZ-Domain Proteins Prevents Hippocampal Long-Term Depression, but Not Long-Term Potentiation.

The group I metabotropic glutamate (mGlu) receptor subtypes, mGlu1 and mGlu5, strongly regulate hippocampal synaptic plasticity. Both harbor PSD-95/discs-large/ZO-1 (PDZ) motifs at their extreme carboxyl terminals, which allow interaction with the PDZ domain of Tamalin, regulate the cell surface expression of group I mGlu receptors, and may modulate their coupling to signaling proteins. We investigated the functional role of this interaction in hippocampal long-term depression (LTD). Acute intracerebral treatment of adult rats with a cell-permeable PDZ-blocking peptide (pep-mGluR-STL), designed to competitively inhibit the interaction between Tamalin and group 1 mGlu receptors, prevented expression of LTD in the hippocampal CA1 region without affecting long-term potentiation (LTP) or basal synaptic transmission. Pep-mGluR-STL prevented facilitation by the group I mGlu receptor agonist, (S)-3,5-Dihydroxyphenylglycine (DHPG), and the mGlu5 agonist, (R,S)-2-chloro-5-Hydroxyphenylglycine (CHPG), of short-term depression (STD) into LTD, suggesting that Tamalin preferentially acts by mediating signaling through mGlu5. These data support that Tamalin is essential for the persistent expression of LTD and that it subserves the effective signaling of group 1 mGlu receptors.

Mitochondrial Quality Control in Aging and Heart Failure: Influence of Ketone Bodies and Mitofusin-Stabilizing Peptides.

Aim: Aging and heart failure (HF) are each characterized by increased mitochondrial damage, which may contribute to further cardiac dysfunction. Mitophagy in response to mitochondrial damage can improve cardiovascular health. HF is also characterized by increased formation and consumption of ketone bodies (KBs), which may activate mitophagy and provide an endogenous mechanism to limit the adverse effects of mitochondrial damage. However, the role of KBs in activation of mitophagy in aging and HF has not been evaluated. Methods: We assessed mitophagy by measuring mitochondrial Parkin accumulation and LC3-mediated autophagosome formation in cardiomyocytes from young (2.5 months), aged (2.5 years), and aged rabbits with HF (2.5 years) induced by aortic insufficiency and stenosis. Levels of reactive oxygen species (ROS) generation and redox balance were monitored using genetically encoded sensors ORP1-roGFP2 and GRX1-roGFP2, targeted to mitochondrial or cytosolic compartments, respectively. Results: Young rabbits exhibited limited mitochondrial Parkin accumulation with small (~1 μm2) puncta. Those small Parkin puncta increased four-fold in aged rabbit hearts, accompanied by elevated LC3-mediated autophagosome formation. HF hearts exhibited fewer small puncta, but many very large Parkin-rich regions (4–5 μm2) with completely depolarized mitochondria. Parkin protein expression was barely detectable in young animals and was much higher in aged and maximal in HF hearts. Expression of mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1) was reduced by almost 50% in HF, consistent with improper fusion-fission, contributing to mitochondrial Parkin build-up. The KB β-hydroxybutyrate (β-OHB) enhanced mitophagy in young and aging myocytes, but not in HF where β-OHB further increased the number of cells with giant Parkin-rich regions. This β-OHB effect on Parkin-rich areas was prevented by cell-permeable TAT-MP1Gly peptide (thought to promote MFN2-dependent fusion). Basal levels of mitochondrial ROS were highest in HF, while cytosolic ROS was highest in aged compared to HF myocytes, suggesting that cytosolic ROS promotes Parkin recruitment to the mitochondria. Conclusion: We conclude that elevated KB levels were beneficial for mitochondrial repair in the aging heart. However, an impaired MFN2-DRP1-mediated fusion-fission process in HF reduced this benefit, as well as Parkin degradation and mitophagic signaling cascade.

The effect of cell‐permeable peptides to limit TRPV‐1 activation during myocardial reperfusion injury

Cardiac reperfusion injury has been implicated in tissue damage caused when blood supply returns after the ischemia. During the reperfusion injury, transient receptor potential channel 1 (TRPV1) mediates cellular responses to stress such as heat, pain, and noxious stimuli. TRPV1 gates calcium and can regulate mitochondrial membrane potential. We hypothesize that the TRPV1 channel and its interaction with calcineurin have a specific role in mediating cardiac cellular injury. To test the hypothesis, adult cardiomyocytes from male Sprague-Dawley rats (225–300grams) were isolated and subjected to hypoxia-reoxygenation for one and two hours, respectively. Isolated adult cardiomyocytes were further treated with cell-permeable peptides and underwent cell death assays such as LDH measurement and Trypan Blue staining to measure cell viability. Moreover, flow cytometry assay was performed to identify the apoptotic rate of cell death upon peptide treatment. According to the results, AnnexinV-FITC/PI double staining demonstrates that cardiomyocytes undergoing hypoxia and reoxygenation (HR) enter the early apoptotic state more frequently than the necrotic state. There is also a significant two-fold difference between normoxia and HR groups in detecting the apoptotic rate. Synthesized peptide against the TRPV1-calcineurin interaction site, V1-cal, maintains a higher level of cell viability when cardiomyocytes were subjected to HR. V1-cal reduces the injury after hypoxia and reoxygenation in primary cardiomyocytes. These data reveal that TRPV1 has a significant role in cardioprotection. On a larger scale, controlling the interaction site between TRPV1 and calcineurin can possibly limit the reperfusion injury. Support or Funding Information The authors would like to thank American Physiological Society (APS) for providing financial support to Z. Zhanzak as a UGREF at Stanford University, School of Medicine, Department of Anesthesiology, Perioperative and Pain Medicine under the supervision of Professor Eric Gross. Annexin V-FITC/PI double staining flow cytometry of primary cardiomyocytes to detect apoptotic rate. LDH assay on primary adult cardiomyocytes Trypan Blue assay on H9C2 cells Immunofluorescence assay on transfected H9C2 cells. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Toward the elucidation of the mechanism for passive membrane permeability of cyclic peptides.

Toward the elucidation of the mechanism for passive membrane permeability of cyclic peptides.

Stealth Peptides Target Cellular Powerhouses to Fight Rare and Common Age-Related Diseases.

Mitochondria are the primary source of energy in most tissues. Mitochondrial dysfunction results in cellular energy deficiency, triggers the production of reactive oxygen species, and initiates various cell death and inflammatory pathways. Several cell-permeable peptides (SS peptides) have been described that selectively target cardiolipin on the inner mitochondrial membrane and promote efficiency of the electron transport chain to produce more ATP. In preclinical disease models, these peptides have been shown to repair damaged mitochondria and promote cellular repair and restore function. By mitigating cell injury, these peptides prevent inflammatory responses that can result in chronic inflammation and tissue remodeling. This peptide technology platform represents a paradigm shift in targeting the fundamental cause of cellular energy failure for age-related degenerative diseases.

Designing Cell-Permeable Macrocyclic Peptides.

Peptides provide an attractive modality for targeting challenging drug targets such as intracellular protein-protein interactions. Unfortunately, peptides are generally impermeable to the cell membrane and inherently susceptible to proteolytic degradation in vivo. Macrocyclization of peptides greatly increases their proteolytic stability and in some cases the cell-penetrating activity. Conjugation of peptidyl cargoes to cyclic cell-penetrating peptides has resulted in potent, cell-permeable, and metabolically stable macrocyclic peptides against intracellular protein targets. Proper conjugation/integration of a peptidyl cargo with a cyclic cell-penetrating peptide is critical to retain the activity of each component and generate a biologically active macrocyclic peptide. This chapter describes the different conjugation strategies that have been developed (including endocyclic, bicyclic, and reversible cyclization methods) and the detailed protocols for their preparation.

Cell death in Leishmania.

Leishmaniases still represent a global scourge and new therapeutic tools are necessary to replace the current expensive, difficult to administer treatments that induce numerous adverse effects and for which resistance is increasingly worrying. In this context, the particularly original organization of the Leishmania parasite in comparison to higher eukaryotes is a great advantage. It allows for the development of new, very specific, and thus non-cytotoxic treatments. Among these originalities, Leishmania cell death can be cited. Despite a classic pattern of apoptosis, key mammalian apoptotic proteins are not present in Leishmania, such as caspases, cell death receptors, and anti-apoptotic molecules. Recent studies have helped to develop a better understanding of parasite cell death, identifying new proteins or even new apoptotic pathways. This review provides an overview of the current knowledge on Leishmania cell death, describing its physiological roles and its phenotype, and discusses the involvement of various proteins: endonuclease G, metacaspase, aquaporin Li-BH3AQP, calpains, cysteine proteinase C, LmjHYD36 and Lmj.22.0600. From these data, potential apoptotic pathways are suggested. This review also offers tools to identify new Leishmania cell death effectors. Lastly, different approaches to use this knowledge for the development of new therapeutic tools are suggested: either inhibition of Leishmania cell death or activation of cell death for instance by treating cells with proteins or peptides involved in parasite death fused to a cell permeant peptide or encapsulated into a lipidic vector to target intra-macrophagic Leishmania cells.

Delivering bioactive cyclic peptides that target Hsp90 as prodrugs

The most challenging issue facing peptide drug development is producing a molecule with optimal physical properties while maintaining target binding affinity. Masking peptides with protecting groups that can be removed inside the cell, produces a cell-permeable peptide, which theoretically can maintain its biological activity. Described are series of prodrugs masked using: (a) O-alkyl, (b) N-alkyl, and (c) acetyl groups, and their binding affinity for Hsp90. Alkyl moieties increased compound permeability, Papp, from 3.3 to 5.6, however alkyls could not be removed by liver microsomes or in-vivo and their presence decreased target binding affinity (IC50 of ≥10 µM). Thus, unlike small molecules, peptide masking groups cannot be predictably removed; their removal is related to the 3-D conformation. O-acetyl groups were cleaved but are labile, increasing challenges during synthesis. Utilising acetyl groups coupled with mono-methylated amines may decrease the polarity of a peptide, while maintaining binding affinity.

HIF-1α-derived cell-penetrating peptides inhibit ERK-dependent activation of HIF-1 and trigger apoptosis of cancer cells under hypoxia.

Hypoxia is frequently encountered in the microenvironment of solid tumors. Hypoxia-inducible factors (HIFs), the main effectors of cell response to hypoxia, promote cancer cell survival and progression. HIF-1α, the oxygen-regulated subunit of HIF-1, is often correlated with oncogenesis and represents an attractive therapeutic target. We have previously reported that activation HIF-1α by ERK involves modification of two serine residues and masking of a nuclear export signal (NES), all inside a 43-amino acid domain termed ERK Targeted Domain (ETD). Overexpression of ETD variants including wild-type, phospho-mimetic (SE) or NES-less (IA) mutant forms caused HIF-1 inactivation in two hepatocarcinoma cell lines, while a phospho-deficient (SA) form was ineffective and acted as a sequence-specific negative control. To deliver these ETD forms directly into cancer cells, they were fused to the HIV TAT-sequence and produced as cell-permeable peptides. When the TAT-ETD peptides were added to the culture medium of Huh7 cells, they entered the cells and, with the exception of ETD-SA, accumulated inside the nucleus, caused mislocalization of endogenous HIF-1α to the cytoplasm, significant reduction of HIF-1 activity and inhibition of expression of specific HIF-1, but not HIF-2, gene targets under hypoxia. More importantly, transduced nuclear TAT-ETD peptides restricted migration, impaired colony formation and triggered apoptotic cell death of cancer cells grown under hypoxia, while they produced no effects in normoxic cells. These data demonstrate the importance of ERK-mediated activation of HIF-1 for low oxygen adaptation and the applicability of ETD peptide derivatives as sequence-specific HIF-1 and cancer cell growth inhibitors under hypoxia.

The Anticancer Activity of a First-in-class Small-molecule Targeting PCNA.

Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is indispensable to cancer cell growth and survival. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was ubiquitously expressed in a broad range of cancer cells and tumor tissues, but not significantly in nonmalignant cells. We found the L126-Y133 region of caPCNA is structurally altered and more accessible to protein-protein interaction. A cell-permeable peptide harboring the L126-Y133 sequence blocked PCNA interaction in cancer cells and selectively kills cancer cells and xenograft tumors. On the basis of these findings, we sought small molecules targeting this peptide region as potential broad-spectrum anticancer agents. By computer modeling and medicinal chemistry targeting a surface pocket partly delineated by the L126-Y133 region of PCNA, we identified a potent PCNA inhibitor (AOH1160) and characterized its therapeutic properties and potential toxicity. AOH1160 selectively kills many types of cancer cells at below micromolar concentrations without causing significant toxicity to a broad range of nonmalignant cells. Mechanistically, AOH1160 interferes with DNA replication, blocks homologous recombination-mediated DNA repair, and causes cell-cycle arrest. It induces apoptosis in cancer cells and sensitizes them to cisplatin treatment. AOH1160 is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it does not cause significant toxicity at 2.5 times of an effective dose. These results demonstrated the favorable therapeutic properties and the potential of AOH1160 as a broad-spectrum therapeutic agent for cancer treatment.

Abstract 101: PHLPP Inhibitory Peptide Administered During CPR Improves Survival After Prolonged Cardiac Arrest

Introduction: Active cooling is more effective than targeted temperature management in improving sudden cardiac arrest (SCA) survival, but can be difficult to implement clinically. We are developing drugs that mimic cooling effects without the need for physical cooling. Cooling protection is mediated by Akt activation. PH domain and Leucine rich repeat Protein Phosphatases (PHLPP) is a phosphatase that negatively regulates Akt. We developed a cell-permeable peptide TAT-PHLPP9c that inhibits PHLPP and reaches heart and brain within 5 min of intravenous administration. Hypothesis: We hypothesize that TAT-PHLPP9c improves outcomes following SCA. This protection involves the improved early metabolic recovery. Methods: C57BL6 mice (n=20) were randomized to receive peptide treatment and appropriate blinding was ensured. A 12 min asystolic arrest was induced with KCl. CPR was started along with TAT-PHLPP9c (7.5 mg/kg) via i.v. Survival was evaluated. Cerebral blood flow (CBF) and metabolic chemical exchange saturation transfer (CEST) contrast were measured with endogenous and dynamic arterial spin labeling (ASL) and CEST MRI respectively. Glucose utilization was assessed by pyruvate dehydrogenase (PDH) phosphorylation and ATP generation. Further, blood taurine and glutamate were measured. Results: Compared to control, TAT-PHLPP9c significantly improved ROSC rates. 3 ROSC mice in the control group died within 10 min while 8 out of 10 ROSC mice in the treatment group survived over 1 h and 4 mice survived to 4 h despite a 12-min arrest. Progressively increased CBF and metabolic CEST contrast were observed in the treated mice compared to controls. p-PDH was decreased by TAT-PHLPP9c (increased activity) at 15 min post-ROSC in both heart and brain. ATP generation was replenished with the peptide treatment. Plasma taurine and glutamate concentrations was reduced by TAT-PHLPP9c as early as 5 min post-ROSC. Further, higher levels of taurine and glutamate were detected in the non-survivors than the survivors. Conclusions: This novel treatment strategy may have a high translational potential to reproduce critical outcomes of CPR cooling without physical cooling.

Abstract 323: A Peptide Inhibitor of PTEN Improves Mouse Survival After Cardiac Arrest

Introduction: Prior works from our laboratory found that cooling protection after cardiac arrest is mediated by enhanced Akt activation and in cardiomyocyte the cooling protection can be reproduced using PTEN chemical inhibitor. The current study extend these works by designing a cell-permeable peptide, TAT-PTEN9c, which is more specific for PTEN. Hypothesis: We hypothesized that TAT-PTEN9c interferes with endogenous PTEN binding to cell membrane adaptor resulting in increased Akt activation, enhanced glucose utilization and improved mouse survival after cardiac arrest. Methods: Mouse cardiomyocytes were isolated from 1-3 day old mouse pups. Western blot was used to determine the efficacy of TAT-PTEN9c for Akt activation. The effect of TAT-PTEN9c on mouse survival after cardiac arrest was determined in a mouse model. TAT-PTEN9c (7.5 mg/kg) was given intravenously (IV) after CPR. As a measure of impaired glucose utilization, sorbitol content in heart and brain was determined by a fluorescence assay of NADH formation using sorbitol dehydrogenase and NAD + . Results: TAT-PTEN9c peptide enhanced Akt activation in mouse cardiomyocytes in a concentration-dependent manner. Akt phosphorylation was observed at 1 μM and further increased with 10 μM TAT-PTEN9c. TAT-PTEN9c blocked the binding of endogenous PTEN to MAGI2 in a co-immunoprecipitation assay, while TAT-PTEN3a control had no inhibitory effect. In a mouse model of cardiac arrest, survival was significantly increased in the TAT-PTEN9c treated group compared to saline controls at 4 h (10/15, 67% vs. 6/15, 40%, P < 0.05) after CPR. TAT-PTEN9c improved MAP at both R30 min and R2h. The treated mice had increased Akt phosphorylation at R15 min in both heart and brain tissues with significantly decreased sorbitol content and reduced release of taurine and glutamate into blood, suggesting improved metabolic recovery and glucose utilization. Conclusion: TAT-PTEN9c can be used after CPR in a mouse SCA model to rapidly enhance Akt activation and decrease glucose shunting to the polyol pathway in critical organs, preventing osmotic injury and early cardiovascular collapse and death.