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.

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.

NRF2 and PPAR-γ Pathways in Oligodendrocyte Progenitors: Focus on ROS Protection, Mitochondrial Biogenesis and Promotion of Cell Differentiation.

An adequate protection from oxidative and inflammatory reactions, together with the promotion of oligodendrocyte progenitor (OP) differentiation, is needed to recover from myelin damage in demyelinating diseases. Mitochondria are targets of inflammatory and oxidative insults and are essential in oligodendrocyte differentiation. It is known that nuclear factor-erythroid 2-related factor/antioxidant responsive element (NRF2/ARE) and peroxisome proliferator-activated receptor gamma/PPAR-γ response element (PPAR-γ/PPRE) pathways control inflammation and overcome mitochondrial impairment. In this study, we analyzed the effects of activators of these pathways on mitochondrial features, protection from inflammatory/mitochondrial insults and cell differentiation in OP cultures, to depict the specificities and similarities of their actions. We used dimethyl-fumarate (DMF) and pioglitazone (pio) as agents activating NRF2 and PPAR-γ, respectively, and two synthetic hybrids acting differently on the NRF2/ARE pathway. Only DMF and compound 1 caused early effects on the mitochondria. Both DMF and pio induced mitochondrial biogenesis but different antioxidant repertoires. Moreover, pio induced OP differentiation more efficiently than DMF. Finally, DMF, pio and compound 1 protected from tumor necrosis factor-alpha (TNF-α) insult, with pio showing faster kinetics of action and compound 1 a higher activity than DMF. In conclusion, NRF2 and PPAR-γ by inducing partially overlapping pathways accomplish complementary functions aimed at the preservation of mitochondrial function, the defense against oxidative stress and the promotion of OP differentiation.

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.

Controlled Iontophoretic Delivery in Vitro and in Vivo of ARN14140-A Multitarget Compound for Alzheimer's Disease.

ARN14140 is a galantamine-memantine conjugate that acts upon both cholinergic and glutamatergic pathways for better management of Alzheimer's disease. Poor oral bioavailability and pharmacokinetics meant that earlier preclinical in vivo studies employed intracerebroventricular injection to administer ARN14140 directly to the brain. The aim of the present study was to evaluate the feasibility of using constant current transdermal iontophoresis for the noninvasive systemic delivery of ARN14140 and to quantify the amounts present in the blood and the brain. Preliminary experiments in vitro were performed using porcine skin and validated with human skin. Cumulative ARN14140 permeation across the skin increased linearly with current density and concentration. Delivery efficiency (i.e., fraction of the amount applied that is delivered) reached an exceptional 76.9%. Statistically equivalent delivery was observed after iontophoresis across human and porcine skin. In vivo studies in male Wistar rats showed that iontophoretic transport of ARN14140 could be controlled using the current density (426.7 ± 42 and 1118.3 ± 73 nmol/cm2 at 0.15 and 0.5 mA/cm2 for 6 h) and demonstrated that transdermal iontophoresis was able to deliver ARN14140 noninvasively to the brain. This is the first report quantifying drug levels in the blood and the brain following transdermal iontophoresis.

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.

Immunomodulators Inspired by Nature: A Review on Curcumin and Echinacea.

The immune system is an efficient integrated network of cellular elements and chemicals developed to preserve the integrity of the organism against external insults and its correct functioning and balance are essential to avoid the occurrence of a great variety of disorders. To date, evidence from literature highlights an increase in immunological diseases and a great attention has been focused on the development of molecules able to modulate the immune response. There is an enormous global demand for new effective therapies and researchers are investigating new fields. One promising strategy is the use of herbal medicines as integrative, complementary and preventive therapy. The active components in medical plants have always been an important source of clinical therapeutics and the study of their molecular pharmacology is an enormous challenge since they offer a great chemical diversity with often multi-pharmacological activity. In this review, we mainly analysed the immunomodulatory/antinflammatory activity of Echinacea spp. and Curcuma longa, focusing on some issues of the phytochemical research and on new possible strategies to obtain novel agents to supplement the present therapies.

Curcumin in Alzheimer's disease: Can we think to new strategies and perspectives for this molecule?

Population aging is an irreversible global trend with economic and socio-political consequences. One of the most invalidating outcomes of aging in the elderly is cognitive decline, leading to dementia and often related to neurodegenerative disorders. Among these latter, Alzheimer's disease (AD) is the major cause of dementia, affecting more than 30 million of individuals worldwide. To date, the treatment of AD remains a challenge because of an incomplete understanding of the events that lead to the selective neurodegeneration typical of Alzheimer's brains. There is an enormous global demand for new effective therapies and researchers are investigating new fields. One promising strategy is the use of nutraceuticals as integrative, complementary and preventive therapy. Curcumin is one example of natural product with anti-AD properties, with promising potential for prevention, treatment and diagnostic. The limitations in the use of curcumin as therapeutic are represented by its pharmacokinetics profile and the low bioavailability after oral administration. However, curcumin has been the focus of intense research for new drug development. Here we analyzed some new approaches that have been applied in the attempt to improve its use, particularly new formulations, changes in the way of administration, nanotechnology-based delivery systems and the hybridization strategy.

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.

Multi-target design strategies in the context of Alzheimer's disease: acetylcholinesterase inhibition and NMDA receptor antagonism as the driving forces.

In recent years, the multi-target-directed ligand concept has been used to design a variety of molecules hitting different biological targets for Alzheimer's disease. We have sought to combine, in the same molecule, the neuroprotective action of N-methyl-D-aspartate receptor antagonism with the symptomatic relief offered by cholinergic activity through acetylcholinesterase inhibition. This strategy could potentially maintain the positive outcomes of memantine-acetylcholinesterase inhibitor combinations, but with the benefits of a single molecule therapy. Herein, we discuss selected examples of multifunctional compounds, which we rationally designed to simultaneously modulate these targets. We also examine the intertwined relationship between acetylcholinesterase, N-methyl-D-aspartate receptors, and other active players in the neurotoxic cascade.

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.

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.

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.

Nature-Inspired Hybrids (NIH) Improve Proteostasis by Activating Nrf2-Mediated Protective Pathways in Retinal Pigment Epithelial Cells.

Antioxidant systems play key roles in many elderly diseases, including age-related macular degeneration (AMD). Oxidative stress, autophagy impairment and inflammation are well-described in AMD, especially in retinal pigment epithelial (RPE) cells. The master regulator of antioxidant defense Nrf2 has been linked to AMD, autophagy and inflammation. In this study, in human ARPE-19 cells, some nature-inspired hybrids (NIH1-3) previously shown to induce Nrf2-mediated protection against oxidative stress were further investigated for their potential against cellular stress caused by dysfunction of protein homeostasis. NIH1-3 compounds increased the expression of two Nrf2-target genes coding defense proteins, HO-1 and SQSTM1/p62, in turn exerting beneficial effects on intracellular redox balance without modification of the autophagy flux. NIH1-3 treatments predisposed ARPE-19 cells to a better response to following exposure to proteasome and autophagy inhibitors, as revealed by the increase in cell survival and decreased secretion of the pro-inflammatory IL-8 compared to NIH-untreated cells. Interestingly, NIH4 compound, through an Nrf2-independent pathway, also increased cell viability and decreased IL-8 secretion, although to a lesser extent than NIH1-3, suggesting that all NIHs are worthy of further investigation into their cytoprotective properties. This study confirms Nrf2 as a valuable pharmacological target in contexts characterized by oxidative stress, such as AMD.

Multitargeted drugs discovery: balancing anti-amyloid and anticholinesterase capacity in a single chemical entity.

Memoquin (1) is a lead compound multitargeted against Alzheimer's disease (AD). It is an AChE inhibitor, free-radical scavenger, and inhibitor of amyloid-ß (Aß) aggregation. A new series of 1 derivatives was designed and synthesized by linking its 2,5-diamino-benzoquinone core with motifs that are present in the structure of known amyloid binding agents like curcumin, the benzofuran derivative SKF64346, or the benzothiazole bearing compounds KHG21834 and BTA-1. The weaker AChE inhibitory potencies and the concomitant nearly equipotent anti-amyloid activities of the new compounds with respect to 1 resulted in a more balanced biological profile against both targets. Selected compounds turned out to be effective Aß aggregation inhibitors in a cell-based assay. By properly combining two or more distinct pharmacological properties in a molecule, we can achieve greater effectiveness compared to single-targeted drugs for investigating AD.

Reversible human serum albumin binding of lipocrine: a circular dichroism study.

Lipocrine has been selected as an effective candidate for in vivo investigation because of its multiple biological properties, namely inhibition of AChE and BChE activities, inhibition of AChE-induced Aß aggregation, and ability to protect cells against reactive oxygen species. To evaluate the possibility for lipocrine to become a lead and to be developed as a multipotent drug for the treatment of Alzheimer's disease, ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters need to be determined. Among ADMET parameters, distribution plays a key role in determining the lead drugability, and the drug binding to plasma proteins greatly influences the drug distribution. Here, the human serum albumin (HSA) binding of lipocrine has been studied by circular dichroism (CD) spectroscopy. The reversible binding of lipocrine is stereoselective as shown by the well-defined induced CD spectrum in its binding to HSA. The intensity of the CD signal changes upon changing the [drug]/[HSA] molar ratio, showing a different behavior for a [drug]/[HSA] up to 2/1 or over this molar ratio, suggesting a binding to multiple sites. Competition experiments show that lipocrine interacts significantly with all the main binding sites on the serum carrier. A direct competition has been monitored for site II and bilirubin-binding site, whereas a noncooperative binding should better describe the displacement observed at site I. Rac-lipocrine and its enantiomers are characterized by two different binding modes. Almost the same induced CD spectra were obtained for both (R)- and (S)-lipocrine complexed to HSA, suggesting a similar stereochemistry for the bound enantiomers.

A Nature-Inspired Nrf2 Activator Protects Retinal Explants from Oxidative Stress and Neurodegeneration.

Oxidative stress (OS) plays a key role in retinal dysfunctions and acts as a major trigger of inflammatory and neurodegenerative processes in several retinal diseases. To prevent OS-induced retinal damage, approaches based on the use of natural compounds are actively investigated. Recently, structural features from curcumin and diallyl sulfide have been combined in a nature-inspired hybrid (NIH1), which has been described to activate transcription nuclear factor erythroid-2-related factor-2 (Nrf2), the master regulator of the antioxidant response, in different cell lines. We tested the antioxidant properties of NIH1 in mouse retinal explants. NIH1 increased Nrf2 nuclear translocation, Nrf2 expression, and both antioxidant enzyme expression and protein levels after 24 h or six days of incubation. Possible toxic effects of NIH1 were excluded since it did not alter the expression of apoptotic or gliotic markers. In OS-treated retinal explants, NIH1 strengthened the antioxidant response inducing a massive and persistent expression of antioxidant enzymes up to six days of incubation. These effects resulted in prevention of the accumulation of reactive oxygen species, of apoptotic cell death, and of gliotic reactivity. Together, these data indicate that a strategy based on NIH1 to counteract OS could be effective for the treatment of retinal diseases.

Synthetic polyamines: an overview of their multiple biological activities.

The binding of polyamines to a variety of receptors and other defined recognition sites has been widely reported. It is well-known that polyamines interact with aspartate, glutamate, and aromatic residues of a given receptor and/or enzyme mainly through the formation of ion bonds, since at physiological pH, protonation of amino groups is nearly complete. From this, the hypothesis arises that a polyamine may be a universal template able to recognize different receptor systems. This hypothesis suggests that both affinity and selectivity may be fine-tuned by inserting appropriate substituents onto the amine functions and by varying the methylene chain lengths between them on the polyamine backbone. In this paper, we detail several application of this design strategy aimed at discovering potent and selective polyamines able to bind neurotransmitter receptors and enzymes, such as muscarinic receptor subtypes, muscle-type nicotinic receptors and acethylcholinesterase.

Functionalized Cannabinoid Subtype 2 Receptor Ligands: Fluorescent, PET, Photochromic and Covalent Molecular Probes.

Cannabinoid subtype 2 receptors (CB2 Rs) are G protein-coupled receptors (GPCRs) belonging to the endocannabinoid system, a complex network of signalling pathways leading to the regulation of key physiological processes. Interestingly, CB2 Rs are strongly up-regulated in pathological conditions correlated with the onset of inflammatory events like cancer and neurodegenerative diseases. Therefore, CB2 Rs represent an important biological target for therapeutic as well as diagnostic purposes. No CB2 R-selective drugs are yet on the market, thus underlining a that deeper comprehension of CB2 Rs' complex activation pathways and their role in the regulation of diseases is needed. Herein, we report an overview of pharmacological and imaging tools such as fluorescent, positron emission tomography (PET), photochromic and covalent selective CB2 R ligands. These molecular probes can be used in vitro as well as in vivo to investigate and explore the unravelled role(s) of CB2 Rs, and they can help to design suitable CB2 R-targeted drugs.

Eye-Light on Age-Related Macular Degeneration: Targeting Nrf2-Pathway as a Novel Therapeutic Strategy for Retinal Pigment Epithelium.

Age-related macular degeneration (AMD) is a common disease with a multifactorial aetiology, still lacking effective and curative therapies. Among the early events triggering AMD is the deterioration of the retinal pigment epithelium (RPE), whose fundamental functions assure good health of the retina. RPE is physiologically exposed to high levels of oxidative stress during its lifespan; thus, the integrity and well-functioning of its antioxidant systems are crucial to maintain RPE homeostasis. Among these defensive systems, the Nrf2-pathway plays a primary role. Literature evidence suggests that, in aged and especially in AMD RPE, there is an imbalance between the increased pro-oxidant stress, and the impaired endogenous detoxifying systems, finally reverberating on RPE functions and survival. In this in vitro study on wild type (WT) and Nrf2-silenced (siNrf2) ARPE-19 cells exposed to various AMD-related noxae (H2O2, 4-HNE, MG132 + Bafilomycin), we show that the Nrf2-pathway activation is a physiological protective stress response, leading downstream to an up-regulation of the Nrf2-targets HO1 and p62, and that a Nrf2 impairment predisposes the cells to a higher vulnerability to stress. In search of new pharmacologically active compounds potentially useful for AMD, four nature-inspired hybrids (NIH) were individually characterized as Nrf2 activators, and their pharmacological activity was investigated in ARPE-19 cells. The Nrf2 activator dimethyl-fumarate (DMF; 10 µM) was used as a positive control. Three out of the four tested NIH (5 µM) display both direct and indirect antioxidant properties, in addition to cytoprotective effects in ARPE-19 cells under pro-oxidant stimuli. The observed pro-survival effects require the presence of Nrf2, with the exception of the lead compound NIH1, able to exert a still significant, albeit lower, protection even in siNrf2 cells, supporting the concept of the existence of both Nrf2-dependent and independent pathways mediating pro-survival effects. In conclusion, by using some pharmacological tools as well as a reference compound, we dissected the role of the Nrf2-pathway in ARPE-19 stress response, suggesting that the Nrf2 induction represents an efficient defensive strategy to prevent the stress-induced damage.