Barbara Monti

Multitarget Drug Discovery for Alzheimer's Disease: Triazinones as BACE‐1 and GSK‐3β Inhibitors

Cumulative evidence strongly supports that the amyloid and tau hypotheses are not mutually exclusive, but concomitantly contribute to neurodegeneration in Alzheimers disease (AD). Thus, the development of multitarget drugs which are involved in both pathways might represent a promising therapeutic strategy. Accordingly, reported here in is the discovery of 6-amino-4-phenyl-3,4-dihydro-1,3,5-triazin-2(1H)-ones as the first class of molecules able to simultaneously modulate BACE-1 and GSK-3β. Notably, one triazinone showed well-balanced in vitro potencies against the two enzymes (IC50 of (18.03±0.01)u2005μM and (14.67±0.78)u2005μM for BACE-1 and GSK-3β, respectively). In cell-based assays, it displayed effective neuroprotective and neurogenic activities and no neurotoxicity. It also showed good brain permeability in a preliminary pharmacokinetic assessment in mice. Overall, triazinones might represent a promising starting point towards high quality lead compounds with an AD-modifying potential.

Novel tacrine-grafted Ugi adducts as multipotent anti-Alzheimer drugs: a synthetic renewal in tacrine-ferulic acid hybrids

Herein we describe the design, multicomponent synthesis, and biological, molecular modeling and ADMET studies, as well as in vitro PAMPA‐blood–brain barrier (BBB) analysis of new tacrine–ferulic acid hybrids (TFAHs). We identified (E)‐3‐(hydroxy‐3‐methoxyphenyl)‐N‐{8[(7‐methoxy‐1,2,3,4‐tetrahydroacridin‐9‐yl)amino]octyl}‐N‐[2‐(naphthalen‐2‐ylamino)2‐oxoethyl]acrylamide (TFAH 10u2009n) as a particularly interesting multipotent compound that shows moderate and completely selective inhibition of human butyrylcholinesterase (IC50=68.2u2005nM), strong antioxidant activity (4.29u2005equiv trolox in an oxygen radical absorbance capacity (ORAC) assay), and good β‐amyloid (Aβ) anti‐aggregation properties (65.6u2009% at 1:1 ratio); moreover, it is able to permeate central nervous system (CNS) tissues, as determined by PAMPA‐BBB assay. Notably, even when tested at very high concentrations, TFAH 10u2009n easily surpasses the other TFAHs in hepatotoxicity profiling (59.4u2009% cell viability at 1000u2005μM), affording good neuroprotection against toxic insults such as Aβ1–40, Aβ1–42, H2O2, and oligomycinu2005A/rotenone on SH‐SY5Y cells, at 1u2005μM. The results reported herein support the development of new multipotent TFAH derivatives as potential drugs for the treatment of Alzheimer′s disease.

3,4-Dihydro-1,3,5-triazin-2(1H)-ones as the First Dual BACE-1/GSK-3β Fragment Hits against Alzheimer’s Disease

One of the main obstacles toward the discovery of effective anti-Alzheimer drugs is the multifactorial nature of its etiopathology. Therefore, the use of multitarget-directed ligands has emerged as particularly suitable. Such ligands, able to modulate different neurodegenerative pathways, for example, amyloid and tau cascades, as well as cognitive and neurogenic functions, are fostered to come. In this respect, we report herein on the first class of BACE-1/GSK-3β dual inhibitors based on a 3,4-dihydro-1,3,5-triazin-2(1H)-one skeleton, whose hit compound 1 showed interesting properties in a preliminary investigation. Notably, compound 2, endowed with well-balanced potencies against the two isolated enzymes (IC50 of 16 and 7 μM against BACE-1 and GSK-3β, respectively), displayed effective neuroprotective and neurogenic activities and no neurotoxicity in cell-based assays. It also showed good brain permeability in a pharmacokinetic assessment in mice. Overall, triazinone derivatives, thanks to the simultaneous modulation of multiple points of the diseased network, might emerge as suitable candidates to be tested in in vivo Alzheimers disease models.

Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer's disease

Multi-target drug discovery is one of the most followed approaches in the active central nervous system (CNS) therapeutic area, especially in the search for new drugs against Alzheimers disease (AD). This is because innovative multi-target-directed ligands (MTDLs) could more adequately address the complexity of this pathological condition. In a continuation of our efforts aimed at a new series of anti-AD MTDLs, we combined the structural features of the cholinesterase inhibitor drug tacrine with that of resveratrol, which is known for its purported antioxidant and anti-neuroinflammatory activities. The most interesting hybrid compounds (5, 8, 9 and 12) inhibited human acetylcholinesterase at micromolar concentrations and effectively modulated Aβ self-aggregation inxa0vitro. In addition, 12 showed intriguing anti-inflammatory and immuno-modulatory properties in neuronal and glial AD cell models. Importantly, the MTDL profile is accompanied by high-predicted blood-brain barrier permeability, and low cytotoxicity on primary neurons.

Changing paradigm to target microglia in neurodegenerative diseases: from anti-inflammatory strategy to active immunomodulation

ABSTRACT Introduction: The importance of microglia in most neurodegenerative pathologies, from Parkinson’s disease to amyotrophic lateral sclerosis and Alzheimer’s disease, is increasingly recognized. Until few years ago, microglial activation in pathological conditions was considered dangerous to neurons due to its causing inflammation. Today we know that these glial cells also play a crucial physiological and neuroprotective role, which is altered in neurodegenerative conditions. Areas covered: The neuroinflammatory hypothesis for neurodegenerative diseases has led to the trial of anti-inflammatory agents as therapeutics with largely disappointing results. New information about the physiopathological role of microglia has highlighted the importance of immunomodulation as a potential new therapeutic approach. This review summarizes knowledge on microglia as a potential therapeutic target in the most common neurodegenerative diseases, with focus on compounds directed toward the modulation of microglial immune response through specific molecular pathways. Expert opinion: Here we support the innovative concept of targeting microglial cells by modulating their activity, rather than simply trying to counteract their inflammatory neurotoxicity, as a potential therapeutic approach for neurodegenerative diseases. The advantage of this therapeutic approach could be to reduce neuroinflammation and toxicity, while at the same time strengthening intrinsic neuroprotective properties of microglia and promoting neuroregeneration.

Nutritional and Pharmacological Strategies to Regulate Microglial Polarization in Cognitive Aging and Alzheimer’s Disease

The study of microglia, the immune cells of the brain, has experienced a renaissance after the discovery of microglia polarization. In fact, the concept that activated microglia can shift into the M1 pro-inflammatory or M2 neuroprotective phenotypes, depending on brain microenvironment, has completely changed the understanding of microglia in brain aging and neurodegenerative diseases. Microglia polarization is particularly important in aging since an increased inflammatory status of body compartments, including the brain, has been reported in elderly people. In addition, inflammatory markers, mainly derived from activated microglia, are widely present in neurodegenerative diseases. Microglial inflammatory dysfunction, also linked to microglial senescence, has been extensively demonstrated and associated with cognitive impairment in neuropathological conditions related to aging. In fact, microglia polarization is known to influence cognitive function and has therefore become a main player in neurodegenerative diseases leading to dementia. As the life span of human beings increases, so does the prevalence of cognitive dysfunction. Thus, therapeutic strategies aimed to modify microglia polarization are currently being developed. Pharmacological approaches able to shift microglia from M1 pro-inflammatory to M2 neuroprotective phenotype are actually being studied, by acting on many different molecular targets, such as glycogen synthase kinase-3 (GSK3) β, AMP-activated protein kinase (AMPK), histone deacetylases (HDACs), etc. Furthermore, nutritional approaches can also modify microglia polarization and, consequently, impact cognitive function. Several bioactive compounds normally present in foods, such as polyphenols, can have anti-inflammatory effects on microglia. Both pharmacological and nutritional approaches seem to be promising, but still need further development. Here we review recent data on these approaches and propose that their combination could have a synergistic effect to counteract cognitive aging impairment and Alzheimer’s disease (AD) through immunomodulation of microglia polarization, i.e., by driving the shift of activated microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype.

Zinc supplementation in rats impairs hippocampal-dependent memory consolidation and dampens post-traumatic recollection of stressful event

Zinc is a trace element important for synaptic plasticity, learning and memory. Zinc deficiency, both during pregnancy and after birth, impairs cognitive performance and, in addition to memory deficits, also results in alterations of attention, activity, neuropsychological behavior and motor development. The effects of zinc supplementation on cognition, particularly in the adult, are less clear. We demonstrate here in adult rats, that 4 week-long zinc supplementation given by drinking water, and approximately doubling normal daily intake, strongly impairs consolidation of hippocampal-dependent memory, tested through contextual fear conditioning and inhibitory avoidance. Furthermore, the same treatment started after memory consolidation of training for the same behavioral tests, substantially dampens the recall of the stressful event occurred 4 weeks before. A molecular correlate of the amnesic effect of zinc supplementation is represented by a dysregulated function of GSK-3ß in the hippocampus, a kinase that participates in memory processes. The possible relevance of these data for humans, in particular regarding post-traumatic stress disorders, is discussed in view of future investigation.

New tacrine dimers with antioxidant linkers as dual drugs: Anti-Alzheimer's and antiproliferative agents

We have designed a series of tacrine-based homo- and heterodimers that incorporate an antioxidant tether (selenoureido, chalcogenide) as new dual compounds: for the treatment of Alzheimers disease and as antiproliferative agents. Symmetrical homodimers bearing a dichalcogenide or selenide-based tether, the best compounds in the series, were found to be strong and highly selective electric eel AChE inhibitors, with inhibition constants within the low nanomolar range. This high inhibitory activity was confirmed on recombinant human AChE for the most interesting derivatives. The three most promising homodimers also showed a good inhibitory activity towards amyloid-β self aggregation. The symmetric disulfide derivative bis[5-(1,2,3,4-tetrahydroacridin-9-ylamino)pentyl]disulfide (19) showed the best multipotent profile and was not neurotoxic on immortalized mouse cortical neurons even at 50xa0μM concentration. These results represent an improvement in activity and selectivity compared to parent tacrine, the first marketed drug against Alzheimers disease. Title compounds also exhibited excellent inxa0vitro antiproliferative activities against a panel of 6 human tumor cell lines, with GI50 values within the submicromolar range for the most potent derivatives (0.12-0.95xa0μM); such values represent a spectacular increase compared to currently-used chemotherapeutic agents, such as 5-FU (up to 306-fold) and cisplatin (up to 162-fold). Cell cycle experiments indicated the accumulation of cells in the G1 phase of the cycle, a different mechanism than the reported for cisplatin. The breast cancer cell lines turned out to be the most sensitive one of the panel tested.

Characterization of DNA methylation as a function of biological complexity via dinucleotide inter-distances

We perform a statistical study of the distances between successive occurrences of a given dinucleotide in the DNA sequence for a number of organisms of different complexity. Our analysis highlights peculiar features of the CG dinucleotide distribution in mammalian DNA, pointing towards a connection with the role of such dinucleotide in DNA methylation. While the CG distributions of mammals exhibit exponential tails with comparable parameters, the picture for the other organisms studied (e.g. fish, insects, bacteria and viruses) is more heterogeneous, possibly because in these organisms DNA methylation has different functional roles. Our analysis suggests that the distribution of the distances between CG dinucleotides provides useful insights into characterizing and classifying organisms in terms of methylation functionalities.

Evidence for purine nucleoside phosphorylase (PNP) release from RAT C6 glioma cells

Intracellular purine turnover is mainly oriented to preserving the level of triphosphate nucleotides, fundamental molecules in vital cell functions that, when released outside cells, act as receptor signals. Conversely, high levels of purine bases and uric acid are found in the extracellular milieu, even in resting conditions. These compounds could derive from nucleosides/bases that, having escaped to cell reuptake, are metabolized by extracellular enzymes similar to the cytosolic ones. Focusing on purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of purine (deoxy)‐nucleosides/bases, we found that it is constitutively released from cultured rat C6 glioma cells into the medium, and has a molecular weight and enzyme activity similar to the cytosolic enzyme. Cell exposure to 10 μM ATP or guanosine triphosphate (GTP) increased the extracellular amount of all corresponding purines without modifying the levels/activity of released PNP, whereas selective activation of ATP P2Y1 or adenosine A2A metabotropic receptors increased PNP release and purine base formation. The reduction to 1% in oxygen supply (2 h) to cells decreased the levels of released PNP, leading to an increased presence of extracellular nucleosides and to a reduced formation of xanthine and uric acid. Conversely, 2 h cell re‐oxygenation enhanced the extracellular amounts of both PNP and purine bases. Thus, hypoxia and re‐oxygenation modulated in opposite manner the PNP release/activity and, thereby, the extracellular formation of purine metabolism end‐products. In conclusion, extracellular PNP and likely other enzymes deputed to purine base metabolism are released from cells, contributing to the purinergic system homeostasis and exhibiting an important pathophysiological role.