Structure-Activity Relationships and Therapeutic Potential of Purinergic P2X7 Receptor Antagonists.

The purinergic P2X7 receptor (P2X7R), an ATP-gated non-selective cation channel, has emerged as a gatekeeper of inflammation that controls the release of proinflammatory cytokines. As a key player in initiating the inflammatory signaling cascade, the P2X7 receptor is currently under intense scrutiny as a target for the treatment of different pathologies, including chronic inflammatory disorders (rheumatoid arthritis and osteoarthritis), chronic neuropathic pain, mood disorders (depression and anxiety), neurodegenerative diseases, ischemia, cancer (leukemia), and many others. For these reasons, pharmaceutical companies have invested in discovering compounds able to modulate the P2X7R and filed many patent applications. This review article presents an account of P2X7R structure, function, and tissue distribution, emphasizing its role in inflammation. Next, we illustrate the different chemical classes of non-competitive P2X7R antagonists reported by highlighting their properties and qualities as clinical candidates for treating inflammatory disorders and neurodegenerative diseases. We also discuss the efforts to develop effective Positron Emission Tomography (PET) radioligands to progress the understanding of the pathomechanisms of neurodegenerative disorders, to provide evidence of drug-target engagement, and to assist clinical dose selection for novel drug therapies.

Amyloid binding and beyond: a new approach for Alzheimer's disease drug discovery targeting Aßo-PrPC binding and downstream pathways.

Amyloid ß oligomers (Aßo) are the main toxic species in Alzheimer's disease, which have been targeted for single drug treatment with very little success. In this work we report a new approach for identifying functional Aßo binding compounds. A tailored library of 971 fluorine containing compounds was selected by a computational method, developed to generate molecular diversity. These compounds were screened for Aßo binding by a combined 19F and STD NMR technique. Six hits were evaluated in three parallel biochemical and functional assays. Two compounds disrupted Aßo binding to its receptor PrPC in HEK293 cells. They reduced the pFyn levels triggered by Aßo treatment in neuroprogenitor cells derived from human induced pluripotent stem cells (hiPSC). Inhibitory effects on pTau production in cortical neurons derived from hiPSC were also observed. These drug-like compounds connect three of the pillars in Alzheimer's disease pathology, i.e. prion, Aß and Tau, affecting three different pathways through specific binding to Aßo and are, indeed, promising candidates for further development.

Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II).

BACKGROUND: Meta-analyses indicated the breakdown of copper homeostasis in the sporadic form of Alzheimer's disease (AD), comprising copper decreases within the brain and copper increases in the blood and the pool not bound to ceruloplasmin (non-Cp Cu, also known in the literature as "free" copper). The calculated non-Cp Cu (Walshe's) index has many limitations. METHODS: A direct fluorescent method for non-Cp Cu detection has been developed and data are presented herein. The study included samples from 147 healthy subjects, 36 stable mild cognitive impairment (MCI) and 89 AD patients, who were tested for non-Cp Cu through the direct method, total serum copper, ceruloplasmin concentration and o-dianisidine ceruloplasmin activity. The indirect non-Cp Cu Walshe's index was also calculated. RESULTS: The direct method was linear (0.9-5.9 µM), precise (within-laboratory coefficient variation of 9.7% for low and 7.1% for high measurements), and had a good recovery. A reference interval (0-1.9 µM) was determined parametrically in 147 healthy controls (27-84 years old). The variation of non-Cp Cu was evaluated according to age and sex. Non-Cp Cu was 1.5 times higher in AD patients (regarding the upper value of the reference interval) than in healthy controls. Healthy, MCI and AD subjects were differentiated through the direct non-Cp Cu method [areas under the curve (AUC)=0.755]. Considering a 95% specificity and a 1.91 µmol/L cut-off, the sensitivity was 48.3% (confidence interval 95%: 38%-58%). The likelihood ratio (LR) was 9.94 for positive test results (LR+) and 0.54 for negative test result (LR-). CONCLUSIONS: The direct fluorescent test reliably and accurately measures non-Cp Cu, thereby determining the probability of having AD.

Non-Ceruloplasmin Copper Distincts Subtypes in Alzheimer's Disease: a Genetic Study of ATP7B Frequency.

Meta-analyses show that serum copper non-bound-to-ceruloplasmin (non-Cp-Cu) is higher in patients with Alzheimer's disease (AD). ATP7B gene variants associate with AD, modulating the size of non-Cp-Cu pool. However, a dedicated genetic study comparing AD patients after stratification for a copper biomarker to demonstrate the existence of a copper subtype of AD has not yet been carried out. An independent patient sample of 287 AD patients was assessed for non-Cp-Cu serum concentrations, rs1801243, rs1061472, and rs732774 ATP7B genetic variants and the APOE4 genotype. Patients were stratified into two groups based on a non-Cp-Cu cutoff (1.9 µM). Single-locus and haplotype-group analyses were performed to define their frequencies in dependence of the non-Cp-Cu group. The two AD subgroups did not differ regarding age, sex, MMSE score, or APOE4 frequency allele, while they did differ regarding non-Cp-Cu concentrations in serum, allele, genotype, and haplotype frequencies of rs1061472 A > G and rs732774 C > T after multiple testing corrections. AD patients with a GG genotype had a 1.76-fold higher risk of having a non-Cp-Cu higher than 1.9 µmol/L (p = 0.029), and those with a TT genotype for rs732774 C > T of 1.8-fold (p = 0.018). After 100,000 permutations for multiple testing corrections, the haplotype containing the AC alleles appeared more frequently in AD patients with normal non-Cp-Cu [43 vs. 33 %; Pm = 0.03], while the haplotype containing the GT risk alleles appeared more frequently in the higher non-Cp-Cu AD (66 vs. 55 %; Pm = 0.01). Genetic heterogeneity sustains a copper AD metabolic subtype; non-Cp-Cu is a marker of this copper AD.

miRNAs for the detection of multidrug resistance: overview and perspectives.

The goal of the present paper is to establish and validate the link between cancer diagnosis and therapy by microRNAs detection. The induction in vitro of some specific microRNAs after treatment with MDR ligands has been outlined. Starting from the results obtained by in vitro induction of MDCK and MDCK-MDR1 cells treated by a MDR1 ligand, a new scenario in the early diagnosis and chemotherapy could be disclosed. To corroborate this perspective a short overview on pancreatic cancer diagnosis and chemotherapeutic treatment has been reported.