Double Attack to Oxidative Stress in Neurodegenerative Disorders: MAO-B and Nrf2 as Elected Targets.

Oxidative stress and neuroinflammation play a pivotal role in triggering the neurodegenerative pathological cascades which characterize neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. In search for potential efficient treatments for these pathologies, that are still considered unmet medical needs, we started from the promising properties of the antidiabetic drug pioglitazone, which has been repositioned as an MAO-B inhibitor, characterized by promising neuroprotective properties. Herein, with the aim to broaden its neuroprotective profile, we tried to enrich pioglitazone with direct and indirect antioxidant properties by hanging polyphenolic and electrophilic features that are able to trigger Nrf2 pathway and the resulting cytoprotective genes' transcription, as well as serve as radical scavengers. After a preliminary screening on MAO-B inhibitory properties, caffeic acid derivative 2 emerged as the best inhibitor for potency and selectivity over MAO-A, characterized by a reversible mechanism of inhibition. Furthermore, the same compound proved to activate Nrf2 pathway by potently increasing Nrf2 nuclear translocation and strongly reducing ROS content, both in physiological and stressed conditions. Although further biological investigations are required to fully clarify its neuroprotective properties, we were able to endow the pioglitazone scaffold with potent antioxidant properties, representing the starting point for potential future pioglitazone-based therapeutics for neurodegenerative disorders.

From Monoamine Oxidase Inhibition to Antiproliferative Activity: New Biological Perspectives for Polyamine Analogs.

Monoamine oxidases (MAOs) are well-known pharmacological targets in neurological and neurodegenerative diseases. However, recent studies have revealed a new role for MAOs in certain types of cancer such as glioblastoma and prostate cancer, in which they have been found overexpressed. This finding is opening new frontiers for MAO inhibitors as potential antiproliferative agents. In light of our previous studies demonstrating how a polyamine scaffold can act as MAO inhibitor, our aim was to search for novel analogs with greater inhibitory potency for human MAOs and possibly with antiproliferative activity. A small in-house library of polyamine analogs (2-7) was selected to investigate the effect of constrained linkers between the inner amine functions of a polyamine backbone on the inhibitory potency. Compounds 4 and 5, characterized by a dianiline (4) or dianilide (5) moiety, emerged as the most potent, reversible, and mainly competitive MAO inhibitors (Ki < 1 µM). Additionally, they exhibited a high antiproliferative activity in the LN-229 human glioblastoma cell line (GI50 < 1 µM). The scaffold of compound 5 could represent a potential starting point for future development of anticancer agents endowed with MAO inhibitory activity.

Galantamine-memantine hybrids for Alzheimer's disease: The influence of linker rigidity in biological activity and pharmacokinetic properties.

Neurodegenerative processes characterizing Alzheimer's disease (AD) are strictly related to the impairment of cholinergic and glutamatergic neurotransmitter systems which provoke synaptic loss. These experimental evidences still represent the foundation of the actual standard-of-care treatment for AD, albeit palliative, consisting on the coadministration of an acetylcholinesterase inhibitor and the NMDAR antagonist memantine. In looking for more effective treatments, we previously developed a series of galantamine-memantine hybrids where compound 1 (ARN14140) emerged with the best-balanced action toward the targets of interest paired to neuroprotective efficacy in a murine AD model. Unfortunately, it showed a suboptimal pharmacokinetic profile, which required intracerebroventricular administration for in vivo studies. In this work we designed and synthesized new hybrids with fewer rotatable bonds, which is related to higher brain exposure. Particularly, compound 2, bearing a double bond in the tether, ameliorated the biological profile of compound 1 in invitro studies, increasing cholinesterases inhibitory potencies and selective antagonism toward excitotoxic-related GluN1/2B NMDAR over beneficial GluN1/2A NMDAR. Furthermore, it showed increased plasma stability and comparable microsomal stability in vitro, paired with lower half-life and faster clearance in vivo. Remarkably, pharmacokinetic evaluations of compound 2 showed a promising increase in brain uptake in comparison to compound 1, representing the starting point for further chemical optimizations.

Polyamine-Drug Conjugates: Do They Boost Drug Activity?

Over the past two decades, the strategy of conjugating polyamine tails with bioactive molecules such as anticancer and antimicrobial agents, as well as antioxidant and neuroprotective scaffolds, has been widely exploited to enhance their pharmacological profile. Polyamine transport is elevated in many pathological conditions, suggesting that the polyamine portion could improve cellular and subcellular uptake of the conjugate via the polyamine transporter system. In this review, we have presented a glimpse on the polyamine conjugate scenario, classified by therapeutic area, of the last decade with the aim of highlighting achievements and fostering future developments.

Inhibition of ß-Amyloid Aggregation in Alzheimer's Disease: The Key Role of (Pro)electrophilic Warheads.

Catechols have been largely investigated as antiaggregating agents toward ß-amyloid peptide. Herein, as a follow up of a previous series of hydroxycinnamic derivatives, we synthesized a small set of dihydroxy isomers for exploring the role of the reciprocal position of the two hydroxyl functions at a molecular level. Para- and ortho-derivatives effectively reduced amyloid fibrillization, while the meta-analogue was devoid of any activity in this respect. Electrochemical analyses showed that the antiaggregating potency correlates with the oxidation potential, hence indicating the proelectrophilic character as a prerequisite for activity. Interestingly, mass spectrometry studies and quantum mechanical calculations revealed different modes of action for active para- and ortho-derivatives, involving covalent or noncovalent interactions with ß-amyloid. The distinctive mode of action is also translated into a different cytotoxicity profile. This work clearly shows how apparently minimal structural modifications can completely change the compound behavior and generate alternative mechanisms of action of proelectrophilic chemical probes.

Bovine Serum Amine Oxidase and Polyamine Analogues: Chemical Synthesis and Biological Evaluation Integrated with Molecular Docking and 3-D QSAR Studies.

Natural polyamines (PAs) are key players in cellular homeostasis by regulating cell growth and proliferation. Several observations highlight that PAs are also implicated in pathways regulating cell death. Indeed, the PA accumulation cytotoxic effect, maximized with the use of bovine serum amine oxidase (BSAO) enzyme, represents a valuable strategy against tumor progression. In the present study, along with the design, synthesis, and biological evaluation of a series of new spermine (Spm) analogues (1-23), a mixed structure-based (SB) and ligand-based (LB) protocol was applied. Binding modes of BSAO-PA modeled complexes led to clarify electrostatic and steric features likely affecting the BSAO-PA biochemical kinetics. LB and SB three-dimensional quantitative structure-activity relationship (Py-CoMFA and Py-ComBinE) models were developed by means of the 3d-qsar.com portal, and their analysis represents a strong basis for future design and synthesis of PA BSAO substrates for potential application in oxidative stress-induced chemotherapy.

Role of Fyn Kinase Inhibitors in Switching Neuroinflammatory Pathways.

Fyn kinase is a member of the Src non-receptor tyrosine kinase family. Fyn is involved in multiple signaling pathways extending from cell proliferation and differentiation to cell adhesion and cell motility, and it has been found to be overexpressed in various types of cancers. In the central nervous system, Fyn exerts several different functions such as axon-glial signal transduction, oligodendrocyte maturation, and myelination, and it is implicated in neuroinflammatory processes. Based on these premises, Fyn emerges as an attractive target in cancer and neurodegenerative disease therapy, particularly Alzheimer's disease (AD), based on its activation by Aß via cellular prion protein and its interaction with tau protein. However, Fyn is also a challenging target since the Fyn inhibitors discovered so far, due to the relevant homology of Fyn with other kinases, suffer from off-target effects. This review covers the efforts performed in the last decade to identify and optimize small molecules that effectively inhibit Fyn, both in enzymatic and in cell assays, including drug repositioning practices, as an opportunity for therapeutic intervention in neurodegeneration.

Novel, Potent, and Druglike Tetrahydroquinazoline Inhibitor That Is Highly Selective for Human Topoisomerase II α over ß.

We disclose a novel class of 6-amino-tetrahydroquinazoline derivatives that inhibit human topoisomerase II (topoII), a validated target of anticancer drugs. In contrast to topoII-targeted drugs currently in clinical use, these compounds do not act as topoII poisons that enhance enzyme-mediated DNA cleavage, a mechanism that is linked to the development of secondary leukemias. Instead, these tetrahydroquinazolines block the topoII function with no evidence of DNA intercalation. We identified a potent lead compound [compound 14 (ARN-21934) IC50 = 2 µM for inhibition of DNA relaxation, as compared to an IC50 = 120 µM for the anticancer drug etoposide] with excellent metabolic stability and solubility. This new compound also shows ~100-fold selectivity for topoIIα over topoß, a broad antiproliferative activity toward cultured human cancer cells, a favorable in vivo pharmacokinetic profile, and the ability to penetrate the blood-brain barrier. Thus, ARN-21934 is a highly promising lead for the development of novel and potentially safer topoII-targeted anticancer drugs.

Memantine Derivatives as Multitarget Agents in Alzheimer's Disease.

Memantine (3,5-dimethyladamantan-1-amine) is an orally active, noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist approved for treatment of moderate-to-severe Alzheimer's disease (AD), a neurodegenerative condition characterized by a progressive cognitive decline. Unfortunately, memantine as well as the other class of drugs licensed for AD treatment acting as acetylcholinesterase inhibitors (AChEIs), provide only symptomatic relief. Thus, the urgent need in AD drug development is for disease-modifying therapies that may require approaching targets from more than one path at once or multiple targets simultaneously. Indeed, increasing evidence suggests that the modulation of a single neurotransmitter system represents a reductive approach to face the complexity of AD. Memantine is viewed as a privileged NMDAR-directed structure, and therefore, represents the driving motif in the design of a variety of multi-target directed ligands (MTDLs). In this review, we present selected examples of small molecules recently designed as MTDLs to contrast AD, by combining in a single entity the amantadine core of memantine with the pharmacophoric features of known neuroprotectants, such as antioxidant agents, AChEIs and Aß-aggregation inhibitors.

Merging memantine and ferulic acid to probe connections between NMDA receptors, oxidative stress and amyloid-ß peptide in Alzheimer's disease.

N-methyl-d-aspartate receptors (NMDAR) are critically involved in the pathogenesis of Alzheimer's disease (AD). Acting as an open-channel blocker, the anti-AD drug memantine preferentially targets NMDAR overactivation, which has been proposed to trigger neurotoxic events mediated by amyloid ß peptide (Aß) and oxidative stress. In this study, we applied a multifunctional approach by conjugating memantine to ferulic acid, which is known to protect the brain from Aß neurotoxicity and neuronal death caused by ROS. The most interesting compound (7) behaved, like memantine, as a voltage-dependent antagonist of NMDAR (IC50 = 6.9 µM). In addition, at 10 µM concentration, 7 exerted antioxidant properties both directly and indirectly through the activation of the Nrf-2 pathway in SH-SY5Y cells. At the same concentration, differently from the parent compounds memantine and ferulic acid alone, it was able to modulate Aß production, as revealed by the observed increase of the non-amyloidogenic sAPPα in H4-SW cells. These findings suggest that compound 7 may represent a promising tool for investigating NMDAR-mediated neurotoxic events involving Aß burden and oxidative damage.

Benextramine and derivatives as novel human monoamine oxidases inhibitors: an integrated approach.

The two human monoamine oxidase isoforms (namely MAO A and MAO B) are enzymes involved in the catabolism of monoamines, including neurotransmitters, and for this reason are well-known and attractive pharmacological targets in neuropsychiatric and neurodegenerative diseases, for which novel pharmacological approaches are necessary. Benextramine is a tetraamine disulfide mainly known as irreversible α-adrenergic antagonist, but able to hit additional targets involved in neurodegeneration. As the molecular structures of monoamine oxidases contain nine cysteine residues, the aim of this study was to evaluate benextramine and eleven structurally related polyamine disulfides as potential MAO inhibitors. Most of the compounds were found to induce irreversible inactivation of MAOs with inactivation potency depending on both the polyamine structure and the enzyme isoform. The more effective compounds generally showed preference for MAO B. Structure-activity relationships studies revealed the key role played by the disulfide core of these molecules in the inactivation mechanism. Docking experiments pointed to Cys323, in MAO A, and Cys172, in MAO B, as target of this type of inhibitors thus suggesting that their covalent binding inside the MAO active site sterically impedes the entrance of substrate towards the FAD cofactor. The effectiveness of benextramine in inactivating MAOs was demonstrated in SH-SY5Y neuroblastoma cell line. These results demonstrated for the first time that benextramine and its derivatives can inactivate human MAOs exploiting a mechanism different from that of the classical MAO inhibitors and could be a starting point for the development of pharmacological tools in neurodegenerative diseases.

Distinct biological responses of metastatic castration resistant prostate cancer cells upon exposure to G-quadruplex interacting naphthalenediimide derivatives.

Small molecules able to bind non-canonical G-quadruplex DNA structures (G4) have been recently tested as novel potential agents for the treatment of prostate cancer thanks to their repression of aberrant androgen receptor gene. However, metastatic castration-resistant prostate cancer (mCRPC), a letal form of prostate cancer, is still incurable. Here we tested two naphthalenediimide derivatives, previously reported as multitarget agents, on a couple of relevant mCRPC cell models (DU145 and PC-3). We showed that these compounds interfere with the RAS/MEK/ERK and PI3K/AKT pathways. Interestingly, both these two biological processes depend upon Epidermal Growth Factor Receptor (EGFR) activation. By means of biological and analytical tools we showed that our compounds are efficient inducers of the structural transition of the EGFR promoter towards a G-quadruplex conformation, ultimately leading to a reduction of the receptor production. The overall result is an interesting cytotoxic profile for these two derivatives. Thanks to their activity at different steps, these compounds can open the way to novel therapeutic approaches for mCRPC that could contribute to escape resistance to selective treatments.

Lights and shadows of electrophile signaling: focus on the Nrf2-Keap1 pathway.

Targeted covalent modification is assuming consolidated importance in drug discovery. In this context, the electrophilic tuning of redox-dependent cell signaling is attracting major interest, as it opens prospect for treating numerous pathologic conditions. Herein, we discuss the rationale and the issues of electrophile-based approaches, focusing on the transcriptional Nrf2-Keap1 pathway as a test case. We also highlight relevant medicinal chemistry strategies researchers have devised to meet the ambitious goal, dwelling on the investigational and therapeutic potential of modulating redox-signaling networks through regulatory cysteine switches.

Exploring the activity of polyamine analogues on polyamine and spermine oxidase: methoctramine, a potent and selective inhibitor of polyamine oxidase.

Fourteen polyamine analogues, asymmetric or symmetric substituted spermine (1-9) or methoctramine (10-14) analogues, were evaluated as potential inhibitors or substrates of two enzymes of the polyamine catabolic pathway, spermine oxidase (SMOX) and acetylpolyamine oxidase (PAOX). Compound 2 turned out to be the best substrate for PAOX, having the highest affinity and catalytic efficiency with respect to its physiological substrates. Methoctramine (10), a well-known muscarinic M2 receptor antagonist, emerged as the most potent competitive PAOX inhibitor known so far (Ki = 10 nM), endowed with very good selectivity compared with SMOX (Ki=1.2 µM vs SMOX). The efficacy of methoctramine in inhibiting PAOX activity was confirmed in the HT22 cell line. Methoctramine is a very promising tool in the design of drugs targeting the polyamine catabolism pathway, both to understand the physio-pathological role of PAOX vs SMOX and for pharmacological applications, being the polyamine pathway involved in various pathologies.

Polyamine-containing etoposide derivatives as poisons of human type II topoisomerases: Differential effects on topoisomerase IIα and IIß.

Etoposide is an anticancer drug that acts by inducing topoisomerase II-mediated DNA cleavage. Despite its wide use, etoposide is associated with some very serious side-effects including the development of treatment-related acute myelogenous leukemias. Etoposide targets both human topoisomerase IIα and IIß. However, the contributions of the two enzyme isoforms to the therapeutic vs. leukemogenic properties of the drug are unclear. In order to develop an etoposide-based drug with specificity for cancer cells that express an active polyamine transport system, the sugar moiety of the drug has been replaced with a polyamine tail. To analyze the effects of this substitution on the specificity of hybrid molecules toward the two enzyme isoforms, we analyzed the activity of a series of etoposide-polyamine hybrids toward human topoisomerase IIα and IIß. All of the compounds displayed an ability to induce enzyme-mediated DNA cleavage that was comparable to or higher than that of etoposide. Relative to the parent drug, the hybrid compounds displayed substantially higher activity toward topoisomerase IIß than IIα. Modeling studies suggest that the enhanced specificity may result from interactions with Gln778 in topoisomerase IIß. The corresponding residue in the α isoform is a methionine.

Novel polyamine-based Histone deacetylases-Lysine demethylase 1 dual binding inhibitors.

Epigenetic modulators Histone deacetylases (HDACs) and Lysine demethylase (LSD1) are validated targets for anticancer therapy. Both HDAC1/2 and LSD1 are found in association with the repressor protein CoREST in a transcriptional co-repressor complex, which is responsible for gene silencing. Combined modulation of both targets results in a synergistic antiproliferative activity. In the present investigation, we report about the design and synthesis of a series of polyamine-based HDACs-LSD1 dual binding inhibitors obtained by coupling Vorinostat and Tranylcypromine. Compound 4 emerged as the most promising of the synthesized series, showing good inhibitory activity towards HDAC1 and LSD1 either in vitro and in cell-based assay (Ki = 42.52 ±â€¯8.94 nM and IC50 = 3.85 µM, respectively). Furthermore, at 70.0 µM compound 4 induced a more pronounced cytotoxic effect than Vorinostat (68.6% vs 56.6% of dead cells) in MCF7 cancer cell line.

Pharmacophore Hybridization To Discover Novel Topoisomerase II Poisons with Promising Antiproliferative Activity.

We used a pharmacophore hybridization strategy to combine key structural elements of merbarone and etoposide and generated new type II topoisomerase (topoII) poisons. This first set of hybrid topoII poisons shows promising antiproliferative activity on human cancer cells, endorsing their further exploration for anticancer drug discovery.

Novel Polyamine-Naphthalene Diimide Conjugates Targeting Histone Deacetylases and DNA for Cancer Phenotype Reprogramming.

A series of hybrid compounds was designed to target histone deacetylases and ds-/G-quadruplex DNAs by merging structural features deriving from Scriptaid and compound 1. Compound 6 binds different DNA arrangements, inhibits HDACs both in vitro and in cells, and is able to induce a reduction of cell proliferation. Moreover, compound 6 displays cell phenotype-reprogramming properties since it prevents the epithelial to mesenchymal transition in cancer cells, inducing a less aggressive and migratory phenotype, which is one of the goals of present innovative strategies in cancer therapies.

Novel xanthone-polyamine conjugates as catalytic inhibitors of human topoisomerase IIα.

It has been proposed that xanthone derivatives with anticancer potential act as topoisomerase II inhibitors because they interfere with the ability of the enzyme to bind its ATP cofactor. In order to further characterize xanthone mechanism and generate compounds with potential as anticancer drugs, we synthesized a series of derivatives in which position 3 was substituted with different polyamine chains. As determined by DNA relaxation and decatenation assays, the resulting compounds are potent topoisomerase IIα inhibitors. Although xanthone derivatives inhibit topoisomerase IIα-catalyzed ATP hydrolysis, mechanistic studies indicate that they do not act at the ATPase site. Rather, they appear to function by blocking the ability of DNA to stimulate ATP hydrolysis. On the basis of activity, competition, and modeling studies, we propose that xanthones interact with the DNA cleavage/ligation active site of topoisomerase IIα and inhibit the catalytic activity of the enzyme by interfering with the DNA strand passage step.

Multitarget drug design strategy in Alzheimer's disease: focus on cholinergic transmission and amyloid-ß aggregation.

AIM: Alzheimer pathogenesis has been associated with a network of processes working simultaneously and synergistically. Over time, much interest has been focused on cholinergic transmission and its mutual interconnections with other active players of the disease. Besides the cholinesterase mainstay, the multifaceted interplay between nicotinic receptors and amyloid is actually considered to have a central role in neuroprotection. Thus, the multitarget drug-design strategy has emerged as a chance to face the disease network. METHODS: By exploiting the multitarget approach, hybrid compounds have been synthesized and studied in vitro and in silico toward selected targets of the cholinergic and amyloidogenic pathways. RESULTS: The new molecules were able to target the cholinergic system, by joining direct nicotinic receptor stimulation to acetylcholinesterase inhibition, and to inhibit amyloid-ß aggregation. CONCLUSION: The compounds emerged as a suitable starting point for a further optimization process.