Didem Dayangac-Erden

Molecular modifications on carboxylic acid derivatives as potent histone deacetylase inhibitors: Activity and docking studies

In the light of known HDAC inhibitors, 33 carboxylic acid derivatives were tested to understand the structural requirements for HDAC inhibition activity. Several modifications were applied to develop the structure-activity relationships of carboxylic acid HDAC inhibitors. HDAC inhibition activities were investigated in vitro by using HeLa nuclear extract in a fluorimetric assay. Molecular docking was also carried out for the human HDAC8 enzyme in order to predict inhibition activity and the 3D poses of inhibitor-enzyme complexes. Of these compounds, caffeic acid derivatives such as chlorogenic acid and curcumin were found to be highly potent compared to sodium butyrate, which is a well-known HDAC inhibitor.

Histone Deacetylase Inhibition Activity and Molecular Docking of (E )‐Resveratrol: Its Therapeutic Potential in Spinal Muscular Atrophy

Spinal muscular atrophy is an autosomal recessive motor neuron disease that is caused by mutation of the survival motor neuron gene (SMN1) but all patients retain a nearly identical copy, SMN2. The disease severity correlates inversely with increased SMN2 copy. Currently, the most promising therapeutic strategy for spinal muscular atrophy is induction of SMN2 gene expression by histone deacetylase inhibitors. Polyphenols are known for protection against oxidative stress and degenerative diseases. Among our candidate prodrug library, we found that (E )‐resveratrol, which is one of the polyphenolic compounds, inhibited histone deacetylase activity in a concentration‐dependent manner and half‐maximum inhibition was observed at 650 μm. Molecular docking studies showed that (E )‐resveratrol had more favorable free energy of binding (−9.09 kcal/mol) and inhibition constant values (0.219 μm) than known inhibitors. To evaluate the effect of (E )‐resveratrol on SMN2 expression, spinal muscular atrophy type I fibroblast cell lines was treated with (E )‐resveratrol. The level of full‐length SMN2 mRNA and protein showed 1.2‐ to 1.3‐fold increase after treatment with 100 μm (E )‐resveratrol in only one cell line. These results indicate that response to (E )‐resveratrol treatment is variable among cell lines. This data demonstrate a novel activity of (E )‐resveratrol and that it could be a promising candidate for the treatment of spinal muscular atrophy.

Carboxylic acid derivatives of histone deacetylase inhibitors induce full length SMN2 transcripts: a promising target for spinal muscular atrophy therapeutics.

Introduction Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder. It is caused by homozygous absence of the survival motor neuron 1 (SMN1) gene. SMN2, which modulates the severity of the disease, represents a major target for therapy. The aim of this study was to investigate whether SMN2 expression can be increased by caffeic acid, chlorogenic acid and curcumin, which are designed by modifications of the carboxylic acid class of histone deacetylase (HDAC) inhibitors. Material and methods Using quantitative real-time PCR, we analysed the levels of full-length SMN2 and Δ7SMN2 mRNA. We performed LDH cytotoxicity assay to analyse whether SMN2 activating concentrations of caffeic acid, chlorogenic acid and curcumin were cytotoxic to fibroblasts. Results We found that caffeic acid and curcumin were more efficient than chlorogenic acid and increased full-length SMN2 mRNA levels 1.5 and 1.7-fold, respectively. Δ7SMN2 mRNA levels were measured to investigate alternative splicing of exon 7. We also found that cytotoxicity was not observed at SMN2 activating concentrations. Conclusions Our data suggest that carboxylic acid derivatives including phenolic structure and symmetry could be a good candidate for SMA treatment.

The relationship between vitamin D receptor gene polymorphisms and bone density, osteocalcin level and growth in adolescents.

ABSTRACT Background/Objective: The relationship between vitamin D receptor gene polymorphisms and bone density, osteocalcin and growth was investigated. Subjects: Eighty eight adolescents aged between 8-15, with no history of illness influencing the level of bone parameters, were examined in our study. Methods: Areal BMD for lumbar spine (L1-4) was assessed by dual energy X-ray absorptiometry (DEXA). Height and weight were measured on the day of the DEXA scans. Serum osteocalcin level was determined by using ELISA method. DNA was extracted from white blood cells, amplified by the polymerase chain reaction (PCR) and the polymorphic sites were analyzed by using ApaI, TaqI and FokI restriction enzymes. Results: The most frequent genotypes were FF (% 54.6), Aa (% 53.4) and Tt (% 48.8). No significant relationship was found between VDR genotypes and areal BMD, osteocalcin level or growth in either sex. But there was a strong tendency for a higher BMD at the lumbar spine of TT and AA genotypes compared to tt and Aa genotypes. The children with TT genotype were taller and heavier than the children with tt genotype. Conclusion: Our results suggest that VDR gene TaqI polymorphism may be associated with body weight and bone mass, but more studies with larger groups should be conducted.

Effects of flavonoid quercetin on survival of motor neuron gene expression

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease that results in muscle weakness and atrophy. To attenuate disease severity, drug development studies have been applied mainly to target the Survival of Motor Neuron 2 (SMN2) gene, which is an important modifier of SMA. Although several compounds have been tested, there is still no cure for SMA. In this study, SMN2‐inducing effects of quercetin, an abundant flavonoid polyphenol in human diet, was investigated in the fibroblast cell lines of two SMA type I patients. Gene expression studies showed that quercetin upregulates SMN2 mRNA up to fourfold, but not the SMN protein level.

A preliminary report on spinal muscular atrophy lymphoblastoid cell lines: Are they an appropriate tool for drug screening?

IntroductionSpinal muscular atrophy (SMA) is a neurodegenerative disease of the motor neurons that results in progressive muscle weakness. It is also the leading hereditary cause of infant mortality. Homozygous loss of the survival motor neuron (SMN1) gene causes SMA, and the number of copies of the SMN2 gene modulates the severity of the disease. Increasing the expression of the SMN2 gene by pharmacological agents is one of the therapeutic approaches currently being implemented.MethodsIn this preliminary study, we investigated the effect of phenylbutyrate, a histone deacetylase (HDAC) inhibitor, on SMN2 expression in two SMA type III Epstein—Barr virus (EBV)-transformed lymphoblastoid cell lines to understand the suitability of lymphoblastoid cell lines in drug screening. These cell lines are regarded as a good source as they can easily be established from the peripheral leucocytes of patients. Quantitative analysis of SMN2 mRNA was performed on established cell lines treated with various concentrations of phenylbutyrate and for a range of incubation periods using real-time polymerase chain reaction. Western blot analysis was used to determine SMN protein levels.ResultsReal-time polymerase chain reaction and Western blot analysis demonstrated that the levels of SMN2 full-length (fl-SMN2) transcripts and protein were not increased in phenylbutyrate-treated cell lines compared to non-treated controls.ConclusionThese results suggest that EBV-transformed lymphoblastoid cell lines are not suitable for studying the effect of certain HDAC inhibitors on SMN2 gene expression.

Transcript levels of plastin 3 and neuritin 1 modifier genes in spinal muscular atrophy siblings

In single gene disorders, patients with the same genotype may have variations in severity. One of the main factors affecting disease severity is modifier genes. Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by degeneration of alpha motor neurons. Plastin 3 (PLS3) is a phenotypic modifier of SMA, and neuritin 1 (NRN1) has also been suggested as a possible modifier gene. The aim of the present study was therefore to analyze PLS3 and NRN1 expression in SMA siblings in four families.

Sepsis: A Challenging Disease With New Promises for Personalized Medicine and Biomarker Discovery

Abstract Sepsis, one of the priority diseases in health research, is a severe disease condition characterized by a systemic inflammatory response to infections, and its incidence still keeps increasing worldwide. To date, no reliable markers for prognosis, early diagnosis, and progression of the disease are available. To answer the question why we do need biomarkers and diagnostic point-of-care systems, we can simply set the frame as follows: to differentiate uncomplicated infection from sepsis, to identify sepsis and organ dysfunction at the earliest stage, to have diagnostic criteria sensitive to the complexity of the patients while retaining the validity and accuracy, and to stratify patients who can benefit from treatment, particularly personalized and specific treatment. This chapter aims to discuss sepsis, mainly in adult population, with its challenges in diagnosis, management, and prognostication from a point of view of promising novel biomarkers in a personalized medicine approach.

Establishment of primary myoblast cell cultures from cryopreserved skeletal muscle biopsies to serve as a tool in related research & development studies

BACKGROUND Primary myoblast cell cultures display the phenotypic characteristics and genetic defects of the donor tissue and represent an in vitro model system reflecting the disease pathology. They have been generated only from freshly harvested tissue biopsies. Here, we describe a novel technique to establish myoblast cell cultures from cryopreserved skeletal muscle biopsy tissues that are useful for diagnostic and research purposes. METHODS AND RESULTS This protocol was performed on seven gradually frozen muscle biopsy specimens from various neuromuscular disorders that were stored in dimethylsulfoxide (DMSO)-supplemented freezing media at -80 °C for up to one year. After storage for varying periods of time, primary myoblast cultures were successfully established from all cryopreserved biopsy tissues without any chromosomal abnormality. Desmin immunoreactivity confirmed that the cell cultures contained >90% pure myoblasts. The myoblasts differentiated into multinucleated myotubes successfully. Furthermore, there were no statistically significant differences in cell viability, metabolic activity, population doubling time, and myocyte enhancer factor 2 (MEF2C) expression between cell cultures established from freshly harvested and one year-stored frozen tissue specimens. CONCLUSIONS This protocol opens up new horizons for basic research and the pre-clinical studies of novel therapies by using cryopreserved skeletal muscle biopsies stored under suitable conditions in tissue banks.

The altered expression of perineuronal net elements during neural differentiation

BackgroundPerineuronal nets (PNNs), which are localized around neurons during development, are specialized forms of neural extracellular matrix with neuroprotective and plasticity-regulating roles. Hyaluronan and proteoglycan link protein 1 (HAPLN1), tenascin-R (TNR) and aggrecan (ACAN) are key elements of PNNs. In diseases characterized by neuritogenesis defects, the expression of these proteins is known to be downregulated, suggesting that PNNs may have a role in neural differentiation.MethodsIn this study, the mRNA and protein levels of HAPLN1, TNR and ACAN were determined and compared at specific time points of neural differentiation. We used PC12 cells as the in vitro model because they reflect this developmental process.ResultsOn day 7, the HAPLN1 mRNA level showed a 2.9-fold increase compared to the non-differentiated state. However, the cellular HAPLN1 protein level showed a decrease, indicating that the protein may have roles in neural differentiation, and may be secreted during the early period of differentiation. By contrast, TNR mRNA and protein levels remained unchanged, and the amount of cellular ACAN protein showed a 3.7-fold increase at day 7. These results suggest that ACAN may be secreted after day 7, possibly due to its large amount of post-translational modifications.ConclusionsOur results provide preliminary data on the expression of PNN elements during neural differentiation. Further investigations will be performed on the role of these elements in neurological disease models.