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International Journal of Molecular... Dec 2019We report on the design, synthesis and molecular modeling study of conjugates of adenosine diphosphate (ADP) and morpholino nucleosides as potential selective...
We report on the design, synthesis and molecular modeling study of conjugates of adenosine diphosphate (ADP) and morpholino nucleosides as potential selective inhibitors of poly(ADP-ribose)polymerases-1, 2 and 3. Sixteen dinucleoside pyrophosphates containing natural heterocyclic bases as well as 5-haloganeted pyrimidines, and mimicking a main substrate of these enzymes, nicotinamide adenine dinucleotide (NAD+)-molecule, have been synthesized in a high yield. Morpholino nucleosides have been tethered to the β-phosphate of ADP via a phosphoester or phosphoramide bond. Screening of the inhibiting properties of these derivatives on the autopoly(ADP-ribosyl)ation of PARP-1 and PARP-2 has shown that the effect depends upon the type of nucleobase as well as on the linkage between ADP and morpholino nucleoside. The 5-iodination of uracil and the introduction of the P-N bond in NAD+-mimetics have shown to increase inhibition properties. Structural modeling suggested that the P-N bond can stabilize the pyrophosphate group in active conformation due to the formation of an intramolecular hydrogen bond. The most active NAD+ analog against PARP-1 contained 5-iodouracil 2'-aminomethylmorpholino nucleoside with IC50 126 ± 6 μM, while in the case of PARP-2 it was adenine 2'-aminomethylmorpholino nucleoside (IC50 63 ± 10 μM). In silico analysis revealed that thymine and uracil-based NAD+ analogs were recognized as the NAD+-analog that targets the nicotinamide binding site. On the contrary, the adenine 2'-aminomethylmorpholino nucleoside-based NAD+ analogs were predicted to identify as PAR-analogs that target the acceptor binding site of PARP-2, representing a novel molecular mechanism for selective PARP inhibition. This discovery opens a new avenue for the rational design of PARP-1/2 specific inhibitors.
Topics: Adenosine Diphosphate; Binding Sites; Humans; Morpholinos; NAD; Niacinamide; Nucleosides; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases
PubMed: 31892271
DOI: 10.3390/ijms21010214 -
British Journal of Cancer Jul 2022Osteosarcoma (OS) is the most common primary bone malignancy. Chemotherapy plays an essential role in OS treatment, potentially doubling 5-year event-free survival if...
BACKGROUND
Osteosarcoma (OS) is the most common primary bone malignancy. Chemotherapy plays an essential role in OS treatment, potentially doubling 5-year event-free survival if tumour necrosis can be stimulated. The canonical Wnt inhibitor Dickkopf-1 (Dkk-1) enhances OS survival in part through upregulation of aldehyde-dehydrogenase-1A1 which neutralises reactive oxygen species originating from nutritional stress and chemotherapeutic challenge.
METHODS
A vivo morpholino (DkkMo) was employed to block the expression of Dkk-1 in OS cells. Cell mitosis, gene expression and bone destruction were measured in vitro and in vivo in the presence and absence of doxorubicin (DRB).
RESULTS
DkkMo reduced the expression of Dkk-1 and Aldh1a1, reduced expansion of OS tumours, preserved bone volume and architecture and stimulated tumour necrosis. This was observed in the presence or absence of DRB.
CONCLUSION
These results indicate that administration of DkkMo with or without chemotherapeutics can substantially improve OS outcome with respect to tumour expansion and osteolytic corruption of bone in experimental OS model.
Topics: Bone Neoplasms; Cell Line, Tumor; Humans; Intercellular Signaling Peptides and Proteins; Morpholinos; Necrosis; Osteosarcoma
PubMed: 35277659
DOI: 10.1038/s41416-022-01764-z -
Scientific Reports Jan 2021Myostatin is a negative regulator of muscle mass and its inhibition represents a promising strategy for the treatment of muscle disorders and type 2 diabetes. However,...
Myostatin is a negative regulator of muscle mass and its inhibition represents a promising strategy for the treatment of muscle disorders and type 2 diabetes. However, there is currently no clinically effective myostatin inhibitor, and therefore novel methods are required. We evaluated the use of antisense phosphorodiamidate morpholino oligomers (PMO) to reduce myostatin expression in skeletal muscle and measured their effects on muscle mass and glucose uptake. C57/Bl6 mice received intramuscular or intravenous injections of anti-myostatin PMOs. Repeated intramuscular administration lead to a reduction in myostatin transcript levels (~ 20-40%), and an increase in muscle mass in chow and high-fat diet (HFD)-fed mice, but insulin-stimulated glucose uptake was reduced in PMO-treated muscles of HFD-fed mice. Five weekly intravenous administrations of 100 nmol PMO did not reduce myostatin expression, and therefore had no significant physiological effects. Unexpectedly, exon skipping levels were higher after intramuscular administration of PMO in HFD- than chow-fed mice. These results suggest that a modest PMO-induced reduction in myostatin transcript levels is sufficient to induce an increase in muscle mass, but that a greater degree of inhibition may be required to improve muscle glucose uptake.
Topics: Animals; Diet, High-Fat; Disease Models, Animal; Exons; Glucose; Glucose Tolerance Test; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Morpholinos; Muscle, Skeletal; Myostatin
PubMed: 33452345
DOI: 10.1038/s41598-021-81222-7 -
Cell and Tissue Research Feb 2021Development of the brain ventricular system of vertebrates and the molecular mechanisms involved are not fully understood. The developmental genes expressed in the...
Development of the brain ventricular system of vertebrates and the molecular mechanisms involved are not fully understood. The developmental genes expressed in the elements of the brain ventricular system such as the ependyma and circumventricular organs act as molecular determinants of cell adhesion critical for the formation of brain ventricular system. They control brain development and function, including the flow of cerebrospinal fluid. Here, we describe the novel distantly related member of the zebrafish L1-CAM family of genes-camel. Whereas its maternal transcripts distributed uniformly, the zygotic transcripts demonstrate clearly defined expression patterns, in particular in the axial structures: floor plate, hypochord, and roof plate. camel expresses in several other cell lineages with access to the brain ventricular system, including the midbrain roof plate, subcommissural organ, organum vasculosum lamina terminalis, median eminence, paraventricular organ, flexural organ, and inter-rhombomeric boundaries. This expression pattern suggests a role of Camel in neural development. Several isoforms of Camel generated by differential splicing of exons encoding the sixth fibronectin type III domain enhance cell adhesion differentially. The antisense oligomer morpholino-mediated loss-of-function of Camel affects cell adhesion and causes hydrocephalus and scoliosis manifested via the tail curled down phenotype. The subcommissural organ's derivative-the Reissner fiber-participates in the flow of cerebrospinal fluid. The Reissner fiber fails to form upon morpholino-mediated Camel loss-of-function. The Camel mRNA-mediated gain-of-function causes the Reissner fiber misdirection. This study revealed a link between Chl1a/Camel and Reissner fiber formation, and this supports the idea that CHL1 is one of the scoliosis factors.
Topics: Animals; Cell Adhesion; Cell Adhesion Molecules; Cerebral Ventricles; Gene Expression Regulation, Developmental; Hydrocephalus; Morpholinos; Phenotype; Phylogeny; Protein Isoforms; RNA, Messenger; Zebrafish; Zebrafish Proteins
PubMed: 32902807
DOI: 10.1007/s00441-020-03270-1 -
Arteriosclerosis, Thrombosis, and... Apr 2015The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although...
OBJECTIVE
The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis.
APPROACH AND RESULTS
Zebrafish fli1a and fli1b mutants were obtained using transposon-mediated gene trap approach. Individual fli1a and fli1b homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older etv2 mutant embryos does not occur in embryos lacking both etv2 and fli1b. Etv2 and fli1b double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast, fli1a mutation did not affect the recovery of etv2 mutant phenotype. Overexpression analyses indicate that both etv2 and fli1b, but not fli1a, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites.
CONCLUSIONS
Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.
Topics: Angiogenic Proteins; Animals; Animals, Genetically Modified; Apoptosis; Binding Sites; Embryo, Nonmammalian; Endothelial Cells; Gene Expression Regulation, Developmental; Genotype; Morpholinos; Mutation; Neovascularization, Physiologic; Phenotype; Promoter Regions, Genetic; Proto-Oncogene Protein c-fli-1; Signal Transduction; Time Factors; Transcription Factors; Transcription, Genetic; Zebrafish; Zebrafish Proteins
PubMed: 25722433
DOI: 10.1161/ATVBAHA.114.304768 -
PloS One 2017NCKX5 is an ion exchanger expressed mostly in pigment cells; however, the functional role for this protein in melanogenesis is not clear. A variant allele of SLC24A5,...
NCKX5 is an ion exchanger expressed mostly in pigment cells; however, the functional role for this protein in melanogenesis is not clear. A variant allele of SLC24A5, the gene encoding NCKX5, has been shown to correlate with lighter skin pigmentation in humans, indicating a key role for SLC24A5 in determining human skin colour. SLC24A5 expression has been found to be elevated in melanoma. Knockdown analyses have shown SLC24A5 to be important for pigmentation, but to date the function of this ion exchanger in melanogenesis has not been fully established. Our data suggest NCKX5 may have an alternative activity that is key to its role in the regulation of pigmentation. Here Xenopus laevis is employed as an in vivo model system to further investigate the function of NCKX5 in pigmentation. SLC24A5 is expressed in the melanophores as they differentiate from the neural crest and develop in the RPE of the eye. Morpholino knockdown and rescue experiments were designed to elucidate key residues and regions of the NCKX5 protein. Unilateral morpholino injection at the 2 cell stage resulted in a reduction of pigmentation in the eye and epidermis of one lateral side of the tadpole. Xenopus and human SLC24A5 can rescue the morpholino effects. Further rescue experiments including the use of ion exchange inactive SLC24A5 constructs raise the possibility that full ion exchanger function of NCKX5 may not be required for rescue of pigmentation.
Topics: Animals; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Morpholinos; Mutation; Phenotype; Skin Pigmentation; Sodium-Calcium Exchanger; Xenopus Proteins; Xenopus laevis
PubMed: 28692664
DOI: 10.1371/journal.pone.0180465 -
Nucleic Acids Research Apr 2020Spinal muscular atrophy (SMA) is a motor neuron disease. Nusinersen, a splice-switching antisense oligonucleotide (ASO), was the first approved drug to treat SMA. Based... (Comparative Study)
Comparative Study
Spinal muscular atrophy (SMA) is a motor neuron disease. Nusinersen, a splice-switching antisense oligonucleotide (ASO), was the first approved drug to treat SMA. Based on prior preclinical studies, both 2'-O-methoxyethyl (MOE) with a phosphorothioate backbone and morpholino with a phosphorodiamidate backbone-with the same or extended target sequence as nusinersen-displayed efficient rescue of SMA mouse models. Here, we compared the therapeutic efficacy of these two modification chemistries in rescue of a severe mouse model using ASO10-29-a 2-nt longer version of nusinersen-via subcutaneous injection. Although both chemistries efficiently corrected SMN2 splicing in various tissues, restored motor function and improved the integrity of neuromuscular junctions, MOE-modified ASO10-29 (MOE10-29) was more efficacious than morpholino-modified ASO10-29 (PMO10-29) at the same molar dose, as seen by longer survival, greater body-weight gain and better preservation of motor neurons. Time-course analysis revealed that MOE10-29 had more persistent effects than PMO10-29. On the other hand, PMO10-29 appears to more readily cross an immature blood-brain barrier following systemic administration, showing more robust initial effects on SMN2 exon 7 inclusion, but less persistence in the central nervous system. We conclude that both modifications can be effective as splice-switching ASOs in the context of SMA and potentially other diseases, and discuss the advantages and disadvantages of each.
Topics: Amides; Animals; Disease Models, Animal; Exons; Humans; Mice, Transgenic; Morpholinos; Motor Activity; Motor Neurons; Muscles; Muscular Atrophy, Spinal; Neuromuscular Junction; Oligonucleotides, Antisense; Phenotype; Phosphoric Acids; RNA Splicing; Spinal Cord; Survival of Motor Neuron 2 Protein; Treatment Outcome
PubMed: 32103257
DOI: 10.1093/nar/gkaa126 -
The International Journal of... Jan 2018Notch signaling plays an essential role in the proliferation, differentiation and cell fate determination of various tissues, including the developing pancreas. One...
Notch signaling plays an essential role in the proliferation, differentiation and cell fate determination of various tissues, including the developing pancreas. One regulator of the Notch pathway is GDE2 (or GDPD5), a transmembrane ecto-phosphodiesterase that cleaves GPI-anchored proteins at the plasma membrane, including a Notch ligand regulator. Here we report that Gdpd5-knockdown in zebrafish embryos leads to developmental defects, particularly, impaired motility and reduced pancreas differentiation, as shown by decreased expression of insulin and other pancreatic markers. Exogenous expression of human GDE2, but not catalytically dead GDE2, similarly leads to developmental defects. Human GDE2 restores insulin expression in Gdpd5a-depleted zebrafish embryos. Importantly, zebrafish Gdpd5 orthologues localize to the plasma membrane where they show catalytic activity against GPI-anchored GPC6. Thus, our data reveal functional conservation between zebrafish Gdpd5 and human GDE2, and suggest that strict regulation of GDE2 expression and catalytic activity is critical for correct embryonic patterning. In particular, our data uncover a role for GDE2 in regulating pancreas differentiation.
Topics: Animals; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Green Fluorescent Proteins; HEK293 Cells; Humans; Isoenzymes; Morpholinos; Organogenesis; Pancreas; Peptide Fragments; Phosphoric Diester Hydrolases; Phylogeny; Protein Domains; RNA, Messenger; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Zebrafish; Zebrafish Proteins
PubMed: 29203233
DOI: 10.1016/j.biocel.2017.11.015 -
Cell Cycle (Georgetown, Tex.) Oct 2019Aneuploidy caused by abnormal chromosome segregation during early embryo development leads to embryonic death or congenital malformation. Centromere protein F (CENPF) is...
Aneuploidy caused by abnormal chromosome segregation during early embryo development leads to embryonic death or congenital malformation. Centromere protein F (CENPF) is a member of centromere protein family that regulates chromosome segregation during mitosis. However, its necessity in early embryo development has not been fully investigated. In this study, expression and function of CENPF was investigated in mouse early embryogenesis. Detection of CENPF expression and localization revealed a cytoplasm, spindle and nuclear membrane related dynamic pattern throughout mitotic progression. Farnesyltransferase inhibitor (FTI) was employed to inhibit CENPF farnesylation in zygotes. The results showed that CENPF degradation was inhibited and its specific localization on nuclear membranes in morula and blastocyst vanished after FTI treatment. Also, CAAX motif mutation leads to failure of CENPF-C630 localization in morula and blastocyst. These results indicate that farnesylation plays a key role during CENPF degradation and localization in early embryos. To further assess CENPF function in parthenogenetic or fertilized embryos development, morpholino (MO) and Trim-Away were used to disturb CENPF function. CENPF knockdown in Metaphase II (MII) oocytes, zygotes or embryos with MO approach resulted in failure to develop into morulae and blastocysts, revealing its indispensable role in both parthenogenetic and fertilized embryos. Disturbing of CENPF with Trim-Away approach in zygotes resulted in impaired development of 2-cell and 4-cell, but did not affect the morula and blastocyst formation because of the recovered expression of CENPF. Taken together, our data suggest CENPF plays an important role during early embryonic development in mice. : CENPF: centromere protein F; MO: morpholino; FTI: Farnesyltransferase inhibitor; CENPE: centromere protein E; IVF: fertilization; MII: metaphase II; SAC: spindle assembly checkpoint; Mad1: mitotic arrest deficient 1; BUB1: budding uninhibited by benzimidazole 1; BUBR1: BUB1 mitotic checkpoint serine/threonine kinase B; Cdc20: cell division cycle 20.
Topics: Animals; Blastocyst; Centromere; Chromosomal Proteins, Non-Histone; Embryo, Mammalian; Embryonic Development; Farnesyltranstransferase; Female; Gene Knockdown Techniques; Metaphase; Mice; Mice, Inbred ICR; Microfilament Proteins; Morpholinos; Morula; Oocytes; Parthenogenesis; Piperidines; Pregnancy; Prenylation; Pyridines; Zygote
PubMed: 31478449
DOI: 10.1080/15384101.2019.1661173 -
Methods in Molecular Biology (Clifton,... 2022Antisense oligonucleotides (AONs) are small synthetic molecules of therapeutic interest for a variety of human disease. Their ability to bind mRNA and affect its...
Antisense oligonucleotides (AONs) are small synthetic molecules of therapeutic interest for a variety of human disease. Their ability to bind mRNA and affect its splicing gives AONs potential use for exon skipping therapies aimed at restoring the dystrophin transcript reading frame for Duchenne muscular dystrophy (DMD) patients. The neutrally charged phosphorodiamidate morpholino oligomers (PMOs) are a stable and relatively nontoxic AON modification. To assess exon skipping efficiency in vitro, it is important to deliver them to target cells. Here, we describe a method for the delivery of PMOs to myoblasts by electroporation. The described protocol for the Amaxa 4D X unit nucleofector system allows efficient processing of 16 samples in one nucleocuvette strip, aiding in high-throughput PMO efficacy screens.
Topics: Dystrophin; Electroporation; Genetic Therapy; Humans; Morpholinos; Muscular Dystrophy, Duchenne; Myoblasts
PubMed: 35213018
DOI: 10.1007/978-1-0716-2010-6_12