Design of Potent and Proteolytically Stable Biaryl-Stapled GLP-1R/GIPR Peptide Dual Agonists.

Recent clinical trials have revealed that the chimeric peptide hormones simultaneously activating glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) demonstrate superior efficacy in glycemic control and body weight reduction, better than those activating the GLP-1R alone. However, the linear peptide-based GLP-1R/GIPR dual agonists are susceptible to proteolytic cleavage by common digestive enzymes present in the gastrointestinal tract and thus not suitable for oral administration. Here, we report the design and synthesis of biaryl-stapled peptides, with and without fatty diacid attachment, that showed potent GLP-1R/GIPR dual agonist activities. Compared to a linear peptide dual agonist and semaglutide, the biaryl-stapled peptides displayed drastically improved proteolytic stability against the common digestive enzymes. Furthermore, two stapled peptides showed excellent efficacy in an oral glucose tolerance test in mice, owing to their potent receptor activity in vitro and good pharmacokinetics exposure upon subcutaneous injection. By exploring a more comprehensive set of biaryl staplers, we expect that this stapling method could facilitate the design of the stapled peptide-based dual agonists suitable for oral administration.

Recombinant Expression and Stapling of a Novel Long-Acting GLP-1R Peptide Agonist.

Owing to their pleiotropic metabolic benefits, glucagon-like peptide-1 receptor (GLP-1R) agonists have been successfully utilized for treating metabolic diseases, such as type 2 diabetes and obesity. As part of our efforts in developing long-acting peptide therapeutics, we have previously reported a peptide engineering strategy that combines peptide side chain stapling with covalent integration of a serum protein-binding motif in a single step. Herein, we have used this strategy to develop a second generation extendin-4 analog rigidified with a symmetrical staple, which exhibits an excellent in vivo efficacy in an animal model of diabetes and obesity. To simplify the scale-up manufacturing of the lead GLP-1R agonist, a semisynthesis protocol was successfully developed, which involves recombinant expression of the linear peptide followed by attachment of a polyethylene glycol (PEG)-fatty acid staple in a subsequent chemical reaction step.

The activity of sulfono-γ-AApeptide helical foldamers that mimic GLP-1.

Existing long α-helix mimicking necessitates the retention of most natural amino acid residues to maintain their biological activity. Here, we report the exploration of helical sulfono-γ-AApeptides with entire unnatural backbones for their ability to structurally and functionally mimic glucagon-like peptide 1 (GLP-1). Our findings suggest that efficient construction of novel GLP-1 receptor (GLP-1R) agonists could be achieved with nanomolar potencies. In addition, the resulting sulfono-γ-AApeptides were also proved to display remarkable stability against enzymatic degradation compared to GLP-1, augmenting their biological potential. This alternative strategy of α-helix mimicking, as a proof of concept, could provide a new paradigm to prepare GLP-1R agonists.

Design and Synthesis of Potent, Long-Acting Lipidated Relaxin-2 Analogs.

Peptide hormone relaxin-2, a member of the insulin family of peptides, plays a key role in hemodynamics and renal function and has shown preclinical efficacy in multiple disease models, including acute heart failure, fibrosis, preeclampsia, and corneal wound healing. Recently, serelaxin, a recombinant version of relaxin-2, has been studied in a large phase 3 clinical trial (RELAX-AHF-2) for acute decompensated heart failure patients with disappointing outcome. The poor in vivo half-life of relaxin-2 may have limited its therapeutic efficacy and long-term cardiovascular benefit. Herein, we have developed a semisynthetic methodology and generated potent, fatty acid-conjugated relaxin analogs with long-acting pharmacokinetic (PK) profile in rodents. The enhanced PK properties translated into improved and long-lasting pharmacodynamic effect in pubic ligament elongation (PLE) studies. The resultant novel relaxin analog, R9-13, represents the first long-acting relaxin-2 analog and could potentially improve the clinical efficacy and outcome for this important peptide hormone. This semisynthetic methodology could also be applied to other cysteine-rich peptides and proteins for half-life extension.

Stapled, Long-Acting Glucagon-like Peptide 2 Analog with Efficacy in Dextran Sodium Sulfate Induced Mouse Colitis Models.

Glucagon-like peptide 2 (GLP-2) is a hormone that has been shown to stimulate intestinal growth and attenuate intestinal inflammation. Despite being efficacious in a variety of animal models of disease, its therapeutic potential is hampered by the short half-life in vivo. We now describe a highly potent, stapled long-acting GLP-2 analog, peptide 10, that has a more than 10-fold longer half-life than teduglutide and improved intestinotrophic and anti-inflammatory effects in mouse models of DSS-induced colitis.

Outcome after Discontinuation of Proton Pump Inhibitors at a Residential Care Site: Quality Improvement Project.

BACKGROUND: Increased prescribing of proton pump inhibitors (PPIs) in the past few decades can be attributed mainly to long-term use of this type of therapy. Recent evidence indicates signals of harm associated with long-term use of PPIs, such as increased risk of Clostridium difficile infection, recurrence of C. difficile infection, and fracture. A few studies have assessed the effectiveness of step-down management of patients receiving long-term PPI therapy in ambulatory care settings. However, it is unknown whether PPIs can be discontinued in older people without return of gastrointestinal symptoms. OBJECTIVES: To determine the proportion of residents receiving long-term PPI therapy who were able to discontinue the drug without experiencing gastrointestinal symptoms warranting recommencement of the PPI or initiation of a histamine-2 receptor antagonist. METHODS: The records of residents who had been taking a PPI for longer than 6 months at a single residential care site were audited by one pharmacist to determine the PPI indication. For residents who fit the criteria for discontinuation (no indication for long-term PPI therapy, not currently experiencing gastrointestinal symptoms, no previous trial of PPI discontinuation without success, and no anxiety when medications are discontinued), the pharmacist faxed a recommendation to discontinue PPI therapy without tapering to the physicians' offices. For cases in which the recommendation was accepted, 3 pharmacists followed the residents weekly for 8 weeks to assess whether gastrointestinal symptoms returned. RESULTS: The pharmacist identified 28 residents who fit the criteria, and the recommendation to discontinue therapy was accepted for 27. At 8 weeks after the intervention, 19 (70%) of these residents were still asymptomatic and did not require re-initiation of medications to manage their gastrointestinal symptoms. CONCLUSIONS: These results support discontinuation of long-term PPI therapy for older people who fit the criteria for discontinuation. The study provided limited evidence to support the use of tapering. However, tapering can be used to identify the lowest effective dose and may increase patient comfort with deprescribing. Further research is needed to determine the effects of and best approaches to PPI discontinuation in older populations.

CONTEXTE: L'augmentation des prescriptions d'inhibiteurs de la pompe à protons (IPP) au cours des dernières décennies peut être attribuée principalement à l'utilisation à long terme de ce type de traitement. Des données récentes indiquent que l'utilisation à long terme des IPP comporte des dangers potentiels, notamment une augmentation du risque d'infection par Clostridium difficile, de récurrence de cette infection et de fracture. Quelques études ont évalué l'efficacité de la déprescription des IPP chez des patients recevant un traitement prolongé en milieu ambulatoire. Cependant, on ne sait pas s'il est possible de cesser l'utilisation d'IPP chez le patient âgé tout en évitant la réapparition de symptômes gastro-intestinaux. OBJECTIF: Déterminer la proportion de résidents pour qui l'on a été en mesure de cesser le traitement à long terme par IPP sans qu'apparaissent des symptômes gastro-intestinaux nécessitant la reprise du traitement par IPP ou l'amorce d'un traitement par antagoniste des récepteurs H2 de l'histamine. MÉTHODES: L'étude a eu lieu dans un seul centre d'hébergement et de soins de longue durée. Un pharmacien y a analysé les dossiers médicaux des résidents qui prenaient des IPP depuis plus de six mois afin de déterminer l'indication du médicament. Pour les résidents répondant aux critères de déprescription (aucune indication pour un traitement d'entretien, aucun symptôme gastro-intestinal à l'heure actuelle, aucune tentative antérieure de déprescrire un IPP en vain et aucune réaction anxieuse à l'arrêt de traitements), le pharmacien a envoyé par télécopieur aux bureaux des médecins un document recommandant l'interruption du traitement par IPP sans posologie dégressive. Dans les cas où la recommandation a été acceptée, trois pharmaciens ont suivi hebdomadairement les résidents pendant huit semaines afin de vérifier si des symptômes gastro-intestinaux réapparaissaient. RÉSULTATS: Le pharmacien a repéré 28 résidents répondant aux critères et la recommandation d'interruption de traitement a été acceptée pour 27 d'entre eux. Huit semaines après l'intervention, 19 (70 %) de ces résidents étaient toujours asymptomatiques et n'ont pas eu besoin qu'on leur prescrive de nouveau des médicaments pour traiter des symptômes gastro-intestinaux. CONCLUSIONS: Ces résultats viennent appuyer l'interruption du traitement à long terme d'IPP chez le patient âgé qui répond aux critères d'interruption. Cette étude n'a founi que peu de preuves qui appuient le recours à la posologie dégressive. Cependant, celle-ci peut servir à déterminer quelle est la plus faible dose efficace et elle peut aider les patients à être plus à l'aise avec l'interruption du traitement. Des recherches plus approfondies sont nécessaires pour préciser les conséquences de l'arrêt des IPP chez les personnes âgées et la meilleure approche à cette fin.

Nuclear factor-erythroid 2-related factor 1 regulates expression of proteasome genes in hepatocytes and protects against endoplasmic reticulum stress and steatosis in mice.

The ubiquitin-proteasome system is important in maintaining protein homeostasis. NFE2-related factor 1 (Nrf1), a transcription factor in the cap 'n' collar basic-leucine zipper family, regulates expression of cytoprotective genes. It was previously shown that liver-specific knockout of Nrf1 (Nrf1LKO) leads to hepatic cell death, steatohepatitis and cancer. However, the mechanisms underlying these pathologies are not clear. Here, we report that Nrf1 is critical for proteasome gene expression in the liver. Liver-specific knockout of Nrf1 results in impaired basal and induced expression of proteasome genes, and diminished proteasome activity in hepatocytes. In addition, our findings demonstrated that endoplasmic reticulum stress signaling pathway was also activated in Nrf1LKO livers. Inhibition of proteasome activity leads to endoplasmic reticulum stress in Nrf1-deficient hepatocytes, prompting the development of steatosis in the liver. Our results indicate that Nrf1 plays an integral role in the maintenance of proteasome function in hepatocytes and in the prevention of liver steatosis development. Moreover, these results highlight an association between proteasome dysfunction, endoplasmic reticulum stress and steatosis.

RNA interference characterization of proteins discovered by proteomic analysis of pancreatic cancer reveals function in cell growth and survival.

OBJECTIVES: There is a clear need for better therapeutics and diagnostics for pancreatic cancer. We aimed to discover plasma membrane-associated proteins overexpressed in pancreatic cancer using quantitative proteomics and apply RNA interference (RNAi) to uncover proteins associated with cancer cell survival. METHODS: Cell surface glycoproteins from 5 pancreatic cancer cell lines were isolated, and differential analyses were performed using mass spectrometry and the "normoid" cell line Hs766T as the comparator. For validation, immunohistochemistry was performed on tissues from 10 independent patients and 2 normal donors. Correlation of protein and mRNA expression level was determined, and functional activity characterized using RNAi. RESULTS: Integrin ß6, CD46, tissue factor, and a novel protein, chromosome 14 open reading frame 1, were identified as overexpressed on pancreatic cancer cell lines. Immunohistochemistry demonstrated the 4 targets were overexpressed in 20% to 70% of primary pancreatic tumor specimens. Small interfering RNA knockdown resulted in a reduction of cellular proliferation by inhibiting DNA synthesis, blocking S-phase progression or induction of apoptosis. CONCLUSIONS: By combining a mass spectrometry identification platform and an RNAi validation platform, we have identified a panel of cell surface glycoproteins that not only are overexpressed, but also play a functional role in pancreatic tumor cell survival.

Transcription factor Nrf1 mediates the proteasome recovery pathway after proteasome inhibition in mammalian cells.

In Saccharomyces cerevisiae, chemical or genetic inhibition of proteasome activity induces new proteasome synthesis promoted by the transcription factor RPN4. This ensures that proteasome activity is matched to demand. This transcriptional feedback loop is conserved in mammals, but its molecular basis is not understood. Here, we report that nuclear factor erythroid-derived 2-related factor 1 (Nrf1), a transcription factor of the cap "n" collar basic leucine zipper family, but not the related Nrf2, is necessary for induced proteasome gene transcription in mouse embryonic fibroblasts (MEFs). Promoter-reporter assays revealed the importance of antioxidant response elements in Nrf1-mediated upregulation of proteasome subunit genes. Nrf1(-/-) MEFs were impaired in the recovery of proteasome activity after transient treatment with the covalent proteasome inhibitor YU101, and knockdown of Nrf1 in human cancer cells enhanced cell killing by YU101. Taken together, our results suggest that Nrf1-mediated proteasome homeostasis could be an attractive target for therapeutic intervention in cancer.

Nrf1 and Nrf2 regulate rat glutamate-cysteine ligase catalytic subunit transcription indirectly via NF-kappaB and AP-1.

Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tert-Butylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidant-responsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null (F1 and F2) fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in F1 and F2 cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-kappaB family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-kappaB binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-kappaB family members.

Liver-specific inactivation of the Nrf1 gene in adult mouse leads to nonalcoholic steatohepatitis and hepatic neoplasia.

Knockout studies have shown that the transcription factor Nrf1 is essential for embryonic development. Nrf1 has been implicated to play a role in mediating activation of oxidative stress response genes through the antioxidant response element (ARE). Because of embryonic lethality in knockout mice, analysis of this function in the adult knockout mouse was not possible. We report here that mice with somatic inactivation of nrf1 in the liver developed hepatic cancer. Before cancer development, mutant livers exhibited steatosis, apoptosis, necrosis, inflammation, and fibrosis. In addition, hepatocytes lacking Nrf1 showed oxidative stress, and gene expression analysis showed decreased expression of various ARE-containing genes, and up-regulation of CYP4A genes. These results suggest that reactive oxygen species generated from CYP4A-mediated fatty acid oxidation work synergistically with diminished expression of ARE-responsive genes to cause oxidative stress in mutant hepatocytes. Thus, Nrf1 has a protective function against oxidative stress and, potentially, a function in lipid homeostasis in the liver. Because the phenotype is similar to nonalcoholic steatohepatitis, these animals may prove useful as a model for investigating molecular mechanisms of nonalcoholic steatohepatitis and liver cancer.

Deficiency of the Nrf1 and Nrf2 transcription factors results in early embryonic lethality and severe oxidative stress.

Nrf1 and Nrf2 are members of the CNC family of bZIP transcription factors that exhibit structural similarities, and they are co-expressed in a wide range of tissues during development. Nrf2 has been shown to be dispensable for growth and development in mice. Nrf2-deficient mice, however, are impaired in oxidative stress defense. We previously showed that loss of Nrf1 function in mice results late gestational embryonic lethality. To determine whether Nrf1 and Nrf2 have overlapping functions during early development and in the oxidative stress response, we generated mice that are deficient in both Nrf1 and Nrf2. In contrast to the late embryonic lethality in Nrf1 mutants, compound Nrf1, Nrf2 mutants die early between embryonic days 9 and 10 and exhibit extensive apoptosis that is not observed in the single mutants. Loss of Nrf1 and Nrf2 leads to marked oxidative stress in cells that is indicated by elevated intracellular reactive oxygen species levels and cell death that is reversed by culturing under reduced oxygen tension or the addition of antioxidants. Compound mutant cells also show increased levels of p53 and induction of Noxa, a death effector p53 target gene, suggesting that cell death is potentially mediated by reactive oxygen species activation of p53. Moreover, we show that expression of genes related to antioxidant defense is severely impaired in compound mutant cells compared with single mutant cells. Together, these findings indicate that the functions of Nrf1 and Nrf2 overlap during early development and to a large extent in regulating antioxidant gene expression in cells.

Generation of a conditionally immortalized myeloid progenitor cell line requiring the presence of both interleukin-3 and stem cell factor to survive and proliferate.

The H-2Kappab temperature-sensitive (ts) A58 transgenic (Immorto) mouse has been used previously to generate conditionally immortalized cells from a number of tissues. The present study aimed to investigate characteristics of primitive myeloid precursor cells derived from H-2Kappab-tsA58 bone marrow. Cell populations were enriched for granulocyte/macrophage progenitors by centrifugal elutriation, and were cultured in the presence and absence of cytokines at the permissive and restrictive temperatures for the A58 oncogene. Cells derived from H-2Kappab-tsA58 mice required both A58 activation and the growth factors, stem cell factor (SCF) and interleukin-3 (IL-3), for long-term cell survival and growth; cells were maintained for > 300 d in culture under these conditions. IL-3- and SCF-dependent clonal cell lines were derived with a phenotype (lin-, Sca-1+, CD34+, ER-MP 58+, ER-MP 12+, ER-MP 20-) characteristic of primitive myeloid progenitors. These cells differentiated on addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) and acquired mature cell morphology with some upregulation of differentiation markers. In conclusion, the A58 oncogene can immortalize haemopoietic progenitor cells. These cells require two cytokines for growth, IL-3 and SCF; as such, they constitute a useful resource for the study of synergistic interactions between growth factors. The ability to develop monocytic cell characteristics also permits the investigation of cytokine-mediated early haemopoietic progenitor cell development.

Nrf1 is critical for redox balance and survival of liver cells during development.

The Nrf1 transcription factor belongs to the CNC subfamily of basic leucine zipper proteins. Knockout of Nrf1 is lethal in mouse embryos, but nothing is known about the cell types that absolutely require its function during development. We show by chimera analysis that Nrf1 is essential for the hepatocyte lineage. Mouse embryonic stem cells lacking Nrf1 developed normally and contributed to most tissues in adult chimeras where Nrf1 is normally expressed. Nrf1-deficient cells contributed to fetal, but not adult, liver cells. Loss of Nrf1 function resulted in liver cell apoptosis in late-gestation chimeric fetuses. Fetal livers from mutant embryos exhibited increased oxidative stress and impaired expression of antioxidant genes, and primary cultures of nrf1(-/-) fetal hepatocytes were sensitive to tert-butyl hydroperoxide-induced cell death, suggesting that impaired antioxidant defense may be responsible for the apoptosis observed in the livers of chimeric mice. In addition, cells deficient in Nrf1 were sensitized to the cytotoxic effects of tumor necrosis factor (TNF). Our results provide in vivo evidence demonstrating an essential role of Nrf1 in the survival of hepatocytes during development. Our results also suggest that Nrf1 may promote cell survival by maintaining redox balance and protecting embryonic hepatocytes from TNF-mediated apoptosis during development.