Claire L. Lill

Global changes in DNA methylation and hydroxymethylation in Alzheimer's disease human brain

DNA methylation (5-methylcytosine [5mC]) is one of several epigenetic markers altered in Alzheimers disease (AD) brain. More recently, attention has been given to DNA hydroxymethylation (5-hydroxymethylcytosine [5hmC]), the oxidized form of 5mC. Whereas 5mC is generally associated with the inhibition of gene expression, 5hmC has been associated with increased gene expression and is involved in cellular processes such as differentiation, development, and aging. Recent findings point toward a role for 5hmC in the development of diseases including AD, potentially opening new pathways for treating AD through correcting methylation and hydroxymethylation alterations. In the present study, levels of 5mC and 5hmC were investigated in the human middle frontal gyrus (MFG) and middle temporal gyrus (MTG) by immunohistochemistry. Immunoreactivity for 5mC and 5hmC were significantly increased in AD MFG (N = 13) and MTG (N = 29) compared with age-matched controls (MFG, N = 13 and MTG, N = 29). Global levels of 5mC and 5hmC positively correlated with each other and with markers of AD including amyloid beta, tau, and ubiquitin loads. Our results showed a global hypermethylation in the AD brain and revealed that levels of 5hmC were also significantly increased in AD MFG and MTG with no apparent influence of gender, age, postmortem delay, or tissue storage time. Using double-fluorescent immunolabeling, we found that in control and AD brains, levels of 5mC and 5hmC were low in astrocytes and microglia but were elevated in neurons. In addition, our colocalization study showed that within the same nuclei, 5mC and 5hmC mostly do not coexist. The present study clearly demonstrates the involvement of 5mC and 5hmC in AD emphasizing the need for future studies determining the exact time frame of these epigenetic changes during the progression of AD pathology.

Synthesis and Biological Evaluation of Novel Analogues of the Pan Class I Phosphatidylinositol 3-Kinase (PI3K) Inhibitor 2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474)

A structure-activity relationship (SAR) study of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) identified substitution at the 4 and 6 positions of the benzimidazole ring as having significant effects on the potency of substituted derivatives. The 6-amino-4-methoxy analogue displayed a greater than 1000-fold potency enhancement over the corresponding 6-aza-4-methoxy analogue against all three class Ia PI 3-kinase enzymes (p110α, p110β, and p110δ) and also displayed significant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo against a U87MG human glioblastoma tumor xenograft model in Rag1(-/-) mice, and at a dose of 50 mg/kg given by ip injection at a qd × 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.

Increased acetyl and total histone levels in post-mortem Alzheimer's disease brain

Histone acetylation is an epigenetic modification that plays a critical role in chromatin remodelling and transcriptional regulation. There is increasing evidence that epigenetic modifications may become compromised in aging and increase susceptibility to the development of neurodegenerative disorders such as Alzheimers disease. Immunohistochemical labelling of free-floating sections from the inferior temporal gyrus (Alzheimers disease, n=14; control, n=17) and paraffin-embedded tissue microarrays containing tissue from the middle temporal gyrus (Alzheimers disease, n=29; control, n=28) demonstrated that acetyl histone H3 and acetyl histone H4 levels, as well as total histone H3 and total histone H4 protein levels, were significantly increased in post-mortem Alzheimers disease brain tissue compared to age- and sex-matched neurologically normal control brain tissue. Changes in acetyl histone levels were proportional to changes in total histone levels. The increase in acetyl histone H3 and H4 was observed in Neuronal N immunopositive pyramidal neurons in Alzheimers disease brain. Using immunolabelling, histone markers correlated significantly with the level of glial fibrillary acidic protein and HLA-DP, -DQ and -DR immunopositive cells and with the pathological hallmarks of Alzheimers disease (hyperphosphorylated tau load and β-amyloid plaques). Given that histone acetylation changes were correlated with changes in total histone protein, it was important to evaluate if protein degradation pathways may be compromised in Alzheimers disease. Consequently, significant positive correlations were also found between ubiquitin load and histone modifications. The relationship between histone acetylation and ubiquitin levels was further investigated in an in vitro model of SK-N-SH cells treated with the proteasome inhibitor Mg132 and the histone deacetylase inhibitor valproic acid. In this model, compromised protein degradation caused by Mg132 lead to elevated histone labelling. In addition, valproic acid worked synergistically with Mg132 in elevating ubiquitin load and causing cell death. These findings highlight important pathological relationships linking a compromise in protein turnover with the histone changes observed in Alzheimers disease post-mortem human brain.

Novel pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors: Exploring the benzenesulfonohydrazide SAR

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.

Isolation and Culture of Adult Human Microglia Within Mixed Glial Cultures for Functional Experimentation and High-Content Analysis

Microglia are thought to be involved in diseases of the adult human brain as well as normal aging processes. While neonatal and rodent microglia are often used in studies investigating microglial function, there are important differences between rodent microglia and their adult human counterparts. Human brain tissue provides a unique and valuable tool for microglial cell and molecular biology. Routine protocols can now enable use of this culture method in many laboratories. Detailed protocols and advice for culture of human brain microglia are provided here. We demonstrate the protocol for culturing human adult microglia within a mixed glial culture and use a phagocytosis assay as an example of the functional studies possible with these cells as well as a high-content analysis method of quantification.

Synthesis and biological evaluation of novel phosphatidylinositol 3-kinase inhibitors: Solubilized 4-substituted benzimidazole analogs of 2-(difluoromethyl)-1-(4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl)- 1H-benzimidazole (ZSTK474)

A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile.

Novel pyrazolo[1,5-a]pyridines as PI3K inhibitors: variation of the central linker group

As part of our investigation into the pyrazolo[1,5-a]pyridines as novel PI3K inhibitors, we report a range of analogues where the central linker portion of the molecule was varied while retaining the pyrazolo[1,5-a]pyridine and arylsulfonyl or arylcarbonyl groups. Isostere generating software BROOD was used to assist with producing ideas. The isoform selectivity of the compounds varied from pan-PI3K for compound 41 to p110α-selective for compound 58 or p110δ-selective for compound 57. The latter two compounds varied only in their sulphur oxidation state.

Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.

Exploring the isoform selectivity of TGX-221 related pyrido[1,2-a]pyrimidinone-based Class IA PI 3-kinase inhibitors: synthesis, biological evaluation and molecular modelling.

A novel series of TGX-221 analogues was prepared and tested for their potency against the p110α, p110β, and p110δ isoforms of the PI3K enzyme, and in two cellular assays. The biological results were interpreted in terms of a p110β comparative model, in order to account for their selectivity towards this isoform. A CH2NH type linker is proposed to allow binding into the specificity pocket proposed to accommodate the high p110β-selectivity of TGX-221, although there was limited steric tolerance for substituents on the pendant ring with the 2-position most favourable for substitution.