Synthetic small-molecule prohormone convertase 2 inhibitors.
Ontology highlight
ABSTRACT:
SUBMITTER: Kowalska D
PROVIDER: S-EPMC2684913 | biostudies-other | 2009 Mar
REPOSITORIES: biostudies-other
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Project description:Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell-derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcsk1 expression were reduced in hypothalami of fasted Snord116 paternal knockout (Snord116p-/m+) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p-/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that Snord116p-/m+ mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH-releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency.
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Project description:Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small molecule furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addition of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochemical cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Molecular modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-Lys-Arg-CH(2)Cl. The compounds presented here are bona fide synthetic small molecule furin inhibitors that exhibit potency in the nanomolar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furin-dependent diseases.
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Project description:Prohormone convertase 2 (PC2) requires interaction with the neuroendocrine protein 7B2 for the production of an activatable zymogen; the mechanism for this effect is unknown. 7B2 could act proactively to generate an activation-competent form of pro-PC2 during synthesis, or block spontaneous generation of activation-incompetent forms. We here demonstrate that addition of exogenous recombinant 7B2 to CHO cells expressing pro-PC2 prevented the unfolding and aggregation of secreted PC2 forms in a dose-dependent manner, as assessed by aggregation assays, activity assays, cross-linking experiments, and sucrose density gradients. Intracellular pro-PC2 was also found to exist in part as higher-order oligomers that were reduced in the presence of coexpressed 7B2. 7B2 addition did not result in the acquisition of enzymatic competence unless added before or very rapidly after pro-PC2 secretion, indicating that an activation-competent structure cannot be maintained in the absence of 7B2. Velocity sedimentation experiments showed that addition of extracellular 7B2 solubilized three different PC2 species from a precipitable aggregate: two activatable pro-PC2 species, the intact zymogen and a zymogen with a partially cleaved propeptide, and an inactive 66-kDa form. Our results suggest that 7B2 possesses chaperone activity that blocks partially unfolded pro-PC2 forms from losing catalytic competence and then aggregating. The loss of the catalytically competent conformer appears to represent the earliest indicator of pro-PC2 unfolding and is followed on a slower time scale by the appearance of aggregates. Because 7B2 expression is not confined to areas expressing pro-PC2, 7B2 may represent a general intracellular and extracellular secretory chaperone.
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Project description:Based upon the observed cleavage of various peptidyl substrates by the recombinant prohormone convertases PC1 and furin, an intramolecularly quenched fluorogenic peptidyl substrate, (o-aminobenzoyl)-Lys-Glu-Arg-Ser-Lys-Arg-Ser-Ala-Leu-Arg-Asp-(3-nitro)Ty r-Ala, was synthesized. In spite of the distance (approx. 33 A) separating the fluorescent donor/acceptor pair, the highly fluorescent o-aminobenzoyl group is efficiently quenched by long-range resonance energy transfer to the (3-nitro)Tyr moiety. Both recombinant human PC1 and human furin recognize and cleave specifically this substrate at the expected Arg-Ser site in a sensitive manner. The Km values for human PC1 and human furin were 17 microM and 30 microM respectively, with Vmax. values of 6.4 microM/h and 18 microM/h. These values differ significantly from those obtained when using a 7-amino-4-methylcoumarin-containing pentapeptidyl substrate where, for similar Km values, the Vmax. values were much lower. The peptide sequence was used to synthesize another peptide incorporating a ketomethylene arginyl pseudopeptide bond. This compound proved to be a potent competitive inhibitor of both human PC1 and human furin, displaying Ki values of 7.2 microM and 2.4 microM respectively.
| S-EPMC1136706 | biostudies-other
Project description:Defective insulin processing is associated with obesity and diabetes. Prohormone convertase 1/3 (PC1/3) is an endopeptidase required for the processing of neurotransmitters and hormones. PC1/3 deficiency and genome-wide association studies relate PC1/3 with early onset obesity. Here, we find that deletion of PC1/3 in obesity-related neuronal cells expressing proopiomelanocortin mildly and transiently change body weight and fail to produce a phenotype when targeted to Agouti-related peptide- or nestin-expressing tissues. In contrast, pancreatic β cell-specific PC1/3 ablation induces hyperphagia with consecutive obesity despite uncontrolled diabetes with glucosuria. Obesity develops not due to impaired pro-islet amyloid polypeptide processing but due to impaired insulin maturation. Proinsulin crosses the blood-brain-barrier but does not induce central satiety. Accordingly, insulin therapy prevents hyperphagia. Further, islet PC1/3 expression levels negatively correlate with body mass index in humans. In this work, we show that impaired PC1/3-mediated proinsulin processing, as observed in human prediabetes, promotes hyperphagic obesity.
| S-EPMC9376086 | biostudies-literature
Project description:Neuropeptides regulate cell-cell signaling and influence many biological processes in vertebrates, including development, growth, and reproduction. The complex processing of neuropeptides from prohormone proteins by prohormone convertases, combined with the evolutionary distance between the chicken and mammalian species that have experienced extensive neuropeptide research, has led to the empirical confirmation of only 18 chicken prohormone proteins. To expand our knowledge of the neuropeptide and prohormone convertase gene complement, we performed an exhaustive survey of the chicken genomic, EST, and proteomic databases using a list of 95 neuropeptide and 7 prohormone convertase genes known in other species. Analysis of the EST resources and 22 microarray studies offered a comprehensive portrait of gene expression across multiple conditions. Five neuropeptide genes (apelin, cocaine-and amphetamine-regulated transcript protein, insulin-like 5, neuropeptide S, and neuropeptide B) previously unknown in chicken were identified and 62 genes were confirmed. Although most neuropeptide gene families known in human are present in chicken, there are several gene not present in the chicken. Conversely, several chicken neuropeptide genes are absent from mammalian species, including C-RF amide, c-type natriuretic peptide 1 precursor, and renal natriuretic peptide. The prohormone convertases, with one exception, were found in the chicken genome. Bioinformatic models used to predict prohormone cleavages confirm that the processing of prohormone proteins into neuropeptides is similar between species. Neuropeptide genes are most frequently expressed in the brain and head, followed by the ovary and small intestine. Microarray analyses revealed that the expression of adrenomedullin, chromogranin-A, augurin, neuromedin-U, platelet-derived growth factor A and D, proenkephalin, relaxin-3, prepronociceptin, and insulin-like growth factor I was most susceptible (P-value<0.005) to changes in developmental stage, gender, and genetic line among other conditions studied. Our complete survey and characterization facilitates understanding of neuropeptides genes in the chicken, an animal of importance to biomedical and agricultural research.
| S-EPMC2814002 | biostudies-literature
Project description:BackgroundThe pig is a biomedical model to study human and livestock traits. Many of these traits are controlled by neuropeptides that result from the cleavage of prohormones by prohormone convertases. Only 45 prohormones have been confirmed in the pig. Sequence homology can be ineffective to annotate prohormone genes in sequenced species like the pig due to the multifactorial nature of the prohormone processing. The goal of this study is to undertake the first complete survey of prohormone and prohormone convertases genes in the pig genome. These genes were functionally annotated based on 35 gene expression microarray experiments. The cleavage sites of prohormone sequences into potentially active neuropeptides were predicted.ResultsWe identified 95 unique prohormone genes, 2 alternative calcitonin-related sequences, 8 prohormone convertases and 1 cleavage facilitator in the pig genome 10.2 assembly and trace archives. Of these, 11 pig prohormone genes have not been reported in the UniProt, UniGene or Gene databases. These genes are intermedin, cortistatin, insulin-like 5, orexigenic neuropeptide QRFP, prokineticin 2, prolactin-releasing peptide, parathyroid hormone 2, urocortin, urocortin 2, urocortin 3, and urotensin 2-related peptide. In addition, a novel neuropeptide S was identified in the pig genome correcting the previously reported pig sequence that is identical to the rabbit sequence. Most differentially expressed prohormone genes were under-expressed in pigs experiencing immune challenge relative to the un-challenged controls, in non-pregnant relative to pregnant sows, in old relative to young embryos, and in non-neural relative to neural tissues. The cleavage prediction based on human sequences had the best performance with a correct classification rate of cleaved and non-cleaved sites of 92% suggesting that the processing of prohormones in pigs is similar to humans. The cleavage prediction models did not find conclusive evidence supporting the production of the bioactive neuropeptides urocortin 2, urocortin 3, torsin family 2 member A, tachykinin 4, islet amyloid polypeptide, and calcitonin receptor-stimulating peptide 2 in the pig.ConclusionsThe present genomic and functional characterization supports the use of the pig as an effective animal model to gain a deeper understanding of prohormones, prohormone convertases and neuropeptides in biomedical and agricultural research.
| S-EPMC3499383 | biostudies-literature
Project description:Prohormone convertase 2 (PC2) functions in the generation of neuropeptides from their precursors. A quantitative peptidomics approach was used to evaluate the role of PC2 in the processing of peptides in a variety of brain regions. Altogether, 115 neuropeptides or other peptides derived from secretory pathway proteins were identified. These peptides arise from 28 distinct secretory pathway proteins, including proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, and many others. Forty one of the peptides found in wild-type (WT) mice were not detectable in any of the brain regions of PC2 knockout mice, and another 24 peptides were present at levels ranging from 20% to 79% of WT levels. Most of the other peptides were not substantially affected by the mutation, with levels ranging from 80% to 120% of WT levels, and only three peptides were found to increase in one or more brain regions of PC2 knockout mice. Taken together, these results are consistent with a broad role for PC2 in neuropeptide processing, but with functional redundancy for many of the cleavages. Comparison of the cleavage sites affected by the absence of PC2 confirms previous suggestions that sequences with a Trp, Tyr, and/or Pro in the P1' or P2' position are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.
| S-EPMC2901178 | biostudies-literature
Project description:BackgroundMutations in WFS1 gene cause Wolfram syndrome, which is a rare autosomal recessive disorder, characterized by diabetes insipidus, diabetes mellitus, optic nerve atrophy, and deafness. The WFS1 gene product wolframin is located in the endoplasmic reticulum. Mice lacking this gene exhibit disturbances in the processing and secretion of peptides, such as vasopressin and insulin. In the brain, high levels of the wolframin protein have been observed in the hippocampus, amygdala, and limbic structures. The aim of this study was to investigate the effect of Wfs1 knockout (KO) on peptide processing in mouse hippocampus. A peptidomic approach was used to characterize individual peptides in the hippocampus of wild-type and Wfs1 KO mice.ResultsWe identified 126 peptides in hippocampal extracts and the levels of 10 peptides differed between Wfs1 KO and wild-type mice at P < 0.05. The peptide with the largest alteration was little-LEN, which level was 25 times higher in the hippocampus of Wfs1 KO mice compared to wild-type mice. Processing (cleavage) of little-LEN from the Pcsk1n gene product proSAAS involves prohormone convertase 2 (PC2). Thus, PC2 activity was measured in extracts prepared from the hippocampus of Wfs1 KO mice. The activity of PC2 in Wfs1 mutant mice was significantly higher (149.9 ± 2.3%, p < 0.0001, n = 8) than in wild-type mice (100.0 ± 7.0%, n = 8). However, Western blot analysis showed that protein levels of 7B2, proPC2 and PC2 were same in both groups, and so were gene expression levels.ConclusionProcessing of proSAAS is altered in the hippocampus of Wfs1-KO mice, which is caused by increased activity of PC2. Increased activity of PC2 in Wfs1 KO mice is not caused by alteration in the levels of PC2 protein. Our results suggest a functional link between Wfs1 and PC2. Thus, the detailed molecular mechanism of the role of Wfs1 in the regulation of PC2 activity needs further investigation.
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Project description:Recent data demonstrate that extracellular signals are transmitted through a network of proteins rather than hierarchical signaling pathways. This network model suggests why inhibition of a single component of a canonical pathway, even when targeting a mutationally activated driver of cancer, has insufficiently dramatic effects on the treatment of cancer. The biological outcome of signals propagated through a network is inherently more robust and resistant to inhibition of a single network component due to compensatory and redundant signaling events. In this study, we performed a functional chemical genetic screen analogous to synthetic lethal screening in yeast genetics to identify novel interactions between signaling inhibitors that would not be predicted based on our current understanding of signaling networks. We screened over 300 drug combinations in nine melanoma cell lines and have identified pairs of compounds that show synergistic cytotoxicity. Among the most robust and surprising results was synergy between sorafenib, a multi-kinase inhibitor with activity against Raf, and diclofenac, a non-steroidal anti-inflammatory drug (NSAID). This synergy did not correlate with the known RAS and BRAF mutational status of the melanoma cell lines. The NSAIDs celecoxib and ibuprofen could qualitatively substitute for diclofenac. Similarly, the MEK inhibitor PD325901 and the Raf inhibitor RAF265 could qualitatively substitute for sorafenib. These drug substitution experiments suggest that inhibition of cyclo-oxygenase and MAP kinase signaling are components of the observed synergistic cytotoxicity. Genome-wide expression profiling demonstrates synergy-specific down-regulation of survival-related genes. This study provides proof of principle that synthetic lethal screening can uncover novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially depending on unexplored components of the cell genotype. RNA from VMM39, DM331, and SLM2 cells with/without mutations in Ras and/or Braf, treated with Sorafenib and/or Diclofenac.
2012-12-06 | E-GEOD-39192 | biostudies-arrayexpress