Multi-target Ligands Research Articles

In silico polypharmacology: retrospective recognition vs. rational design.

The „one drug – one target – one disease“ paradigm in drug discovery has been reconsidered during the last decade. This paradigm change was mainly caused by high attrition rates in drug approvals due to toxicity and lack of efficacy. Computational techniques play an important role in prediction and recognition of novel targets for approved drugs. We will discuss two machine learning approaches – self organizing maps and inverse distance weighting – for polypharmacological profiling of bioactive compounds, exemplified by two prospective studies [1,2]. While the recognition of occasional polypharmacological behavior is an established task, the rational design of multitarget ligands remains challenging. Dual or multi-target ligands have several advantages compared with selective compounds, including improved efficacy and more simple pharmacokinetic and pharmacodynamic properties in comparison to the combination of several drugs. In this context we present two in silico approaches to design dual inhibitors of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH). The first study contains the discovery of a benzimidazole-based dual 5-LO/sEH inhibitor by means of in silico screening [3]. The strategy of the virtual screening protocol was an exhaustive pairwise evaluation of pharmacophore models for both targets to obtain a dual-target pharmacophore model. Our second study deals with the development of a fragment based strategy for dual-target drug discovery. Here, we applied a modified self-organizing map algorithm for in silico recognition of molecular fragments binding both targets. The predicted properties were confirmed by complementary screening techniques: STD-NMR and enzyme assay. The enlargement of the fragment hit led to submicromolar dual target inhibitor of sEH and 5-LO.[4]

Porphyrin‐Based Design of Bioinspired Multitarget Quadruplex Ligands

Secondary nucleic acid structures, such as DNA and RNA quadruplexes, are potential targets for cancer therapies. Ligands that interact with these targets could thus find application as anticancer agents. Synthetic G-quartets have recently found numerous applications, including use as bioinspired G-quadruplex ligands. Herein, the design, synthesis and preliminary biophysical evaluation of a new prototype multitarget G-quadruplex ligand, (PNA)PorphySQ, are reported, where peptidic nucleic acid guanine ((PNA)G) was incorporated in the porphyrin-templated synthetic G-quartet (PorphySQ). Using fluorescence resonance energy transfer (FRET)-melting experiments, PorphySQ was shown to possess enhanced quadruplex-interacting properties thanks to the presence of four positively charged (PNA)G residues that improve its electrostatic interactions with the binding site of both DNA and RNA quadruplexes (i.e., their negatively charged and accessible G-quartets), thereby making (PNA)PorphySQ an interesting prototype of a multitarget ligand. Both the chemical stability and water solubility of (PNA)PorphySQ are improved over the non-PNA derivative (PorphySQ), which are desirable properties for drug development, and while improvements remain to be made, this ligand is a promising lead for the further development of multitarget G-quadruplex ligands.

From Single Target to Multitarget/Network Therapeutics in Alzheimer’s Therapy

Brain network dysfunction in Alzheimer’s disease (AD) involves many proteins (enzymes), processes and pathways, which overlap and influence one another in AD pathogenesis. This complexity challenges the dominant paradigm in drug discovery or a single-target drug for a single mechanism. Although this paradigm has achieved considerable success in some particular diseases, it has failed to provide effective approaches to AD therapy. Network medicines may offer alternative hope for effective treatment of AD and other complex diseases. In contrast to the single-target drug approach, network medicines employ a holistic approach to restore network dysfunction by simultaneously targeting key components in disease networks. In this paper, we explore several drugs either in the clinic or under development for AD therapy in term of their design strategies, diverse mechanisms of action and disease-modifying potential. These drugs act as multi-target ligands and may serve as leads for further development as network medicines.

Exploring the chemical space of multitarget ligands using aligned self-organizing maps.

Design of multitarget drugs and polypharmacological compounds has become popular during the past decade. However, the main approach to design such compounds is to link two selective ligands via a flexible linker. Although such chimeric ligands often have reasonable potency in vitro, the in vivo efficacy is low due to high molecular weight, low ligand efficiency, and poor pharmacokinetic profile. We developed an unprecedented in silico approach for fragment-based design of multitarget ligands. It relies on superposition of the chemical spaces related to the affinity on single targets represented by self-organizing maps. We used this approach for screening of molecular fragments, which bind to the enzymes 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH). Using STD-NMR and activity-based assays, we were able to identify fragments binding to both targets. Furthermore, we were able to expand one of the fragments to a potent dual inhibitor bearing a reasonable molecular weight (MW = 446) and high affinity to both targets (IC50 of 0.03 μM toward 5-LO and 0.17 μM toward sEH).

Inhibitors of the Arachidonic Acid Cascade: Interfering with Multiple Pathways

Modulators of the arachidonic acid cascade have been in the focus of research for treatments of inflammation and pain for several decades. Targeting this complex pathway experiences a paradigm change towards the design and development of multi-target inhibitors, exhibiting improved efficacy and less undesired side effects. This minireview summarizes recent developments in the field of designed multi-target ligands of the arachidonic acid cascade. In addition to the well-known dual inhibitors of 5-lipoxygenase and cyclooxygenase-2 such as licofelone, very recent developments are discussed. Especially, multi-target inhibitors interfering with the cytochrome P450 pathway via inhibition of soluble epoxide hydrolase seem to offer a novel opportunity for development of novel anti-inflammatory drugs.

Quinones bearing non-steroidal anti-inflammatory fragments as multitarget ligands for Alzheimer’s disease

The anti-amyloid properties shared by several quinones inspired the design of a new series of hybrids derived from the multi-target drug candidate memoquin (1). The hybrids consist of a central benzoquinone core and a fragment taken from non-steroidal anti-inflammatory drugs, connected through polyamine linkers. The new hybrids retain the potent anti-aggregating activity of the parent 1, while exhibiting micromolar AChE inhibitory activities. Remarkably, 2, 4, (R)-6 and (S)-6 were Aβ aggregation inhibitors even more potent than 1. The balanced amyloid/cholinesterase inhibitory profile is an added value that makes the present series of compounds promising leads against Alzheimer’s disease.

Nuclear Estrogen Receptors Co-activation Mechanisms

Estrogens play very important role in opening the transcription event, which is a final step of activation of the first order mediators as receptors or channels in the cell wall by information coming from the outside of the cell. For the long time the exact step by step mechanism of cellular transfer of information to the cell nuclei was not known. Currently many new informations are available. Very important seems the step of phosphorylation and therefore desensitization of the target proteins. All peptide kinases, especially serine and threonine, like protein kinases A and C, RAS and MAP kinases, cycline kinases are potential or confirmed biological targets. Except them elements of the transcription complexes like p160.SRC-1, histon acetyltransferase and histon deacetylase, CBP/p300, TRAP/DRIP, NSD1, PPARγ/PGC-1, NCOR1, SMRT, REA were also found useful. Finally estrogens are able to activate other receptors, namely aryl hydrocarbon receptors (AhR) and estrogen receptor related proteins (ERR). It is well known that many types of cancer are related to the direct or indirect excessive activation of nuclear estrogen receptors, therefore their inhibition could be crucial in many estrogen-related cancers. Understanding the interactions in such complexes would help in developing new and better multi-target cures and finding new ligands with better pharmacological and pharmacokinetic properties.

Design and synthesis of 6,7-dimethoxyquinazoline analogs as multi-targeted ligands for α1- and AII-receptors antagonism

Multiple-targeted ligands can have certain advantages for the management of hypertension which has multiple controls. Molecules with dual bioactivities are available in literature for treating metabolic disorders like diabetes, hypertension and hypercholesterolemia. After scrutinizing the SAR of prazosin-type α1-blockers and AII-antagonists it was planned to develop dual α1- and AII-antagonists. Five series of quinazoline derivatives were synthesized and evaluated as dual α1- and AII-antagonists on rat aortic strips for the blockade of known α1- and AII-agonist mediated contractions. Many compounds showed balanced activity on both the receptors but compound (22) was found to be the most active derivative having higher antagonistic activity on both the receptors. In the in vivo experiments the chosen compound (22) was slightly less active than prazosin but was found to be equipotent to losartan. These findings shed a new light on the structural requirements for both α1- as well as AII-receptor antagonists.

A Novel Multi-Target Drug Screening Strategy Directed Against Key Proteins of DAPk Family

Screening and identifying multi-target ligands becomes a daunting task when there are very few matching pharmacophoric features among the proteins. Herein, we describe a novel screening strategy to identify multi-target ligands for proteins having varying pharmacophoric features with their ligands. This strategy was adopted to identify multi-target ligands for death-associated protein kinase (DAPk) family. The role of the kinase activity of DAPk in eukaryotic cell apoptosis and the ability of bioavailable DAPk inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In this work, we employed a novel strategy using the existing computational approaches to design multi-target inhibitors, which can potentially inhibit one or any combination of the three DAPk family members. The strategy employs a combination of merged pharmacophore matching, database screening and molecular docking to reliably identify potential multi-target inhibitors targeted against DAPk protein family.

The multitarget opioid ligand LP1's effects in persistent pain and in primary cell neuronal cultures

Persistent pain states, such as those caused by nerve injury or inflammation, are associated with altered sensations, allodynia and hyperalgesia, that are resistant to traditional analgesics. A contribution to development and maintenance in altered pain perception comes from nociceptive processing and descending modulation from supraspinal sites. A multitarget ligand seems to be useful for pain relief with a decreased risk of adverse events and a considerable analgesic efficacy. The multitarget MOR agonist–DOR antagonist LP1, (3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benazocin-3(2H)-yl]-N-phenylpropanamide, is a central acting antinociceptive agent with low potential to induce tolerance. LP1 was tested in models of neuropathic pain – induced by chronic constriction injury (CCI) of the left sciatic nerve – and inflammatory pain – produced by intraplantar injection of carrageenan. In CCI rats, subcutaneous (s.c.) LP1 (3 mg/kg) showed a significant antiallodynic effect, measured with von Frey filaments, and antihyperalgesic effect, evoked in response to a radiant heat stimulus with plantar test. Analogously, LP1 significantly reduced allodynic and hyperalgesic thresholds in a model of inflammatory pain induced by carrageenan. To evaluate the contribution of opioid receptor subtypes in LP1 antinociceptive effects, the multitarget LP1 profile was assessed using selective opioid antagonists. Moreover, functional electrophysiological in vitro assays, using primary cortical and spinal cord networks, allowed to define the “pharmacological fingerprint” of LP1. The EC50 values in this functional screening seem to confirm LP1 as a potent opioid ligand (EC50 = 0.35 fM and EC50 = 44 pM in spinal cord and frontal cortex, respectively). Using a NeuroProof data-base of well characterised reference compounds, a similarity profile of LP1 to opioid and non-opioid drugs involved in pain modulation was detected. Our studies seem to support that multitarget ligand approach should be useful for persistent pain conditions in which mechanical allodynia and thermal hyperalgesia are significant components of the nociceptive response.

Application of Structural Interaction Fingerpints (SIFts) into post-docking analysis - insight into activity and selectivity

Cheminformatic methods, such as Virtual Screening, constitute a vital part of modern drug design process. This technique enables not only viable prediction of physicochemical properties of the molecules, but also effective database mining, being particularly useful tool in search for ligands of desired activity. Successful performance in case of single target drugs, implies a potential to extend its capabilities to compounds bearing desired activity towards multiple receptors. In this research, we present application of Structural Interaction Fingerprints (SIFts) [1] combined with Machine Learning (ML) as a method to select single- and multi-target ligands from the docking results. A handful of protein kinases pairs was designated as targets. Collection of pseudo selective compounds, with various activity profiles, was acquired from ChEMBL database. Furthermore, for each target a set of ligands of various activity was aggregated. Decoy structures were random ligands from ZINC database. The compounds were docked into respective proteins, and SIFts were calculated for each protein-ligand complex. Training sets used in ML experiments consisted of cluster centroids of active and inactive ligands, whereas test sets were composed of remaining compounds, that returned docking poses. The key aim of this study is to develop a viable method to filter the docking results, so that the compounds meeting desired activity profile are selected.

Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

Background and MethodologyRecently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach.Principal FindingsA functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity.Conclusions and SignificanceOverall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Novel multitarget ligand ITH33/IQM9.21 provides neuroprotection in in vitro and in vivo models related to brain ischemia

ITH33/IQM9.21 is a novel compound belonging to a family of glutamic acid derivatives, synthesized under the hypothesis implying that multitarget ligands may provide more efficient neuroprotection than single-targeted compounds. In rat hippocampal slices, oxygen plus glucose deprivation followed by re-oxygenation (OGD/Reox) elicited 42% cell death. At 1 μM, ITH33/IQM9.21 mitigated this damage by 26% and by 55% at 3 μM. OGD/Reox also elicited mitochondrial depolarization, overproduction of reactive oxygen species (ROS), enhanced expression of nitric oxide synthase (iNOS) and reduction of GSH levels. These changes were almost fully prevented when 3 μM ITH33/IQM9.21 was present during slice treatment with OGD/Reox. In isolated hippocampal neurons, ITH33/IQM9.21 reduced [Ca2+]c transients induced by a high K+ depolarizing solution or glutamate. In a photothrombotic model of stroke in mice, intraperitoneal injection of ITH33/IQM9.21 at 1.25 mg/kg, 2.5 mg/kg or 5 mg/kg given before and during 2 days after stroke induction, reduced infarct volume by over 45%. Furthermore, when the compound was administered 1 h post-stroke, a similar effect was observed. In conclusion, these in vitro and in vivo results suggest that ITH33/IQM9.21 exhibits neuroprotective effects to protect the vulnerable neurons at the ischemic penumbra by an effective and multifaceted mechanism, mediated by reduction of Ca2+ overload, providing mitochondrial protection and antioxidant actions.

New insights into pharmacological profile of LASSBio-579, a multi-target N-phenylpiperazine derivative active on animal models of schizophrenia

Previous behavioral and receptor binding studies on N-phenylpiperazine derivatives by our group indicated that LASSBio-579, LASSBio-580 and LASSBio-581 could be potential antipsychotic lead compounds. The present study identified LASSBio-579 as the most promising among the three compounds, since it was the only one that inhibited apomorphine-induced climbing (5mg/kg p.o.) and apomorphine-induced hypothermia (15mg/kg p.o.). Furthermore, LASSBio-579 (0.5mg/kg p.o.) was effective in the ketamine-induced hyperlocomotion test and prevented the prepulse inhibition deficits induced by apomorphine, DOI and ketamine with different potencies (1mg/kg, 0.5mg/kg and 5mg/kg p.o., respectively). LASSBio-579 also induced a motor impairment, catalepsy and a mild sedative effect but only at doses 3–120 times higher than those with antipsychotic-like effects. In addition, LASSBio-579 (0.5 and 1mg/kg p.o.) reversed the catalepsy induced by WAY 100,635, corroborating its action on both dopaminergic and serotonergic neurotransmission and pointing to the contribution of 5-HT1A receptor activation to its pharmacological profile. Moreover, co-administration of sub-effective doses of LASSBio-579 with sub-effective doses of clozapine or haloperidol prevented the apomorphine-induced climbing without induction of catalepsy. In summary, our results characterize LASSBio-579 as a multi-target ligand active in pharmacological animal models of schizophrenia, confirming that this compound could be included in development programs aiming at a new drug for treating schizophrenia.

In the Rush for Green Gold: Can Green Tea Delay Age-Progressive Brain Neurodegeneration?

It is evident that brain aging engages changes in biological systems linked to synaptic function and cell metabolism and in the capacity to cope with different stresses that are either idiopathic in nature, or subject to environmental insults. In a substantial segment of the aging population there is a pathological transition to cognitive and behavioral dysfunction and thus, age constitutes the primary risk factor for Alzheimer's disease and other neurodegenerative disorders. To address the etiological complexity of aging and age-associated conditions, a new paradigm gaining increasing acceptance considers the use of multi-targeted ligands or combination of drugs to modulate several targets at once. During the past years intensive efforts are dedicated to the implementation of life style habits such as exercise and dietary compounds/supplements in combination with symptomatic treatment drugs to improve age-related cognitive decline and to attenuate motor and neurological dysfunction in neurodegenerative diseases. The catechin polyphenols constituents of green tea, which were for long time regarded merely as dietary antioxidants, have caught our and other scientist's attention because of their diverse pharmacological activities, which have been allied to a possible beneficial action on brain health. This review will elaborate on the impact of nutritional supplementation on brain function in general, and provide a compilation of the most updated literature on epidemiology, clinical and animal studies with green tea polyphenols in age-associated cognitive decline and in fighting neurodegenerative diseases. To conclude, a future perspective on the utility and assigned patents with green tea constituents will be presented.

Design of dual ligands using excessive pharmacophore query alignment

Dual- or multi-target ligands have gained increased attention in the past years due to several advantages, including more simple pharmacokinetic and phamarcodynamic properties compared to a combined application of several drugs. Furthermore multi-target ligands often possess improved efficacy [1]. We present a new approach for the discovery of dual-target ligands using aligned pharmacophore models combined with a shape-based scoring. Starting with two sets of known active compounds for each target, a number of different pharmacophore models is generated and subjected to pairwise graph-based alignment using the Kabsch-Algorithm [2,3]. Since a compound may be able to bind to different targets in different conformations, the algorithm aligns pairs of pharmacophore models sharing the same features which are not necessarily at the exactly same spatial distance. Using the aligned models, a pharmacophore search on a multi-conformation-database is performed to find compounds matching both models. The potentially “dual” ligands are scored by a shape-based comparison with the known active molecules using ShaEP [4]. Using this approach, we performed a prospective fragment-based virtual screening for dual 5-LO/sEH inhibitors. Both enzymes play an important role in the arachidonic acid cascade and are involved in inflammatory processes, pain, cardiovascular diseases and allergic reactions [5,6]. Beside several new selective inhibitors we were able to find a compound inhibiting both enzymes in low micromolar concentrations. The results indicate that the idea of aligned pharmacophore models can be successfully employed for the discovery of dual-target ligands.

Fragment-based identification of multi-target ligands by self-organizing map alignment

In the recent years the prevalent paradigm in drug discovery of „one drug – one target – one disease“, following the assumption that highly selective ligands would avoid unwandted side effects caused by binding to seconday non-theratpeutic targets, got reconsidered. The results of post-genomic and network biology showed that proteins rarely act in isolated systems but rather as a part of a highly connected network [1]. It was further shown that the efficacy of several approved drugs is traced back to the fact that they act on multiple targets [2]. Therefore inhibiting a single target of such a network might not lead to the desired therapeutic effect. These findings lead to a shift towards polypharmacology [3] and the rational design of selective multi-target drugs, which have often improved efficacy [4]. But the design of multi target drugs is still a great challenge in regard of a sufficient activity on each target as well as an adequate pharmacokinetic profile [5]. Early design strategies tried to link the pharmacophors of known inhibitors, however these methods often lead to high molecular weight and low ligand efficiency. We present a new approach based on self-organizing maps [3,6] (SOM) for the identification of multi-target fragments. We describe a workflow that initially identifies multi-target relevant substructures with a combination of maximum common substructure search and the alignment of multiple SOMs. Furthermore, these substructures are trained together with a fragment library on additional SOMs to find new multi-target fragments, validated by saturation transfer difference (STD)-NMR and biochemical assay systems. We used our approach for the identification of new dual-acting inhibitors of 5Lipoxygenase (5-LO) and soluble Epoxide Hydrolase (sEH), both enzymes located in the arachidonic acid cascade and involved in inflammatory processes, pain and cadiovascular diseases.

Molecular determinants of magnolol targeting both RXRα and PPARγ.

Nuclear receptors retinoic X receptor α (RXRα) and peroxisome proliferator activated receptor γ (PPARγ) function potently in metabolic diseases, and are both important targets for anti-diabetic drugs. Coactivation of RXRα and PPARγ is believed to synergize their effects on glucose and lipid metabolism. Here we identify the natural product magnolol as a dual agonist targeting both RXRα and PPARγ. Magnolol was previously reported to enhance adipocyte differentiation and glucose uptake, ameliorate blood glucose level and prevent development of diabetic nephropathy. Although magnolol can bind and activate both of these two nuclear receptors, the transactivation assays indicate that magnolol exhibits biased agonism on the transcription of PPAR-response element (PPRE) mediated by RXRα:PPARγ heterodimer, instead of RXR-response element (RXRE) mediated by RXRα:RXRα homodimer. To further elucidate the molecular basis for magnolol agonism, we determine both the co-crystal structures of RXRα and PPARγ ligand-binding domains (LBDs) with magnolol. Structural analyses reveal that magnolol adopts its two 5-allyl-2-hydroxyphenyl moieties occupying the acidic and hydrophobic cavities of RXRα L-shaped ligand-binding pocket, respectively. While, two magnolol molecules cooperatively accommodate into PPARγ Y-shaped ligand-binding pocket. Based on these two complex structures, the key interactions for magnolol activating RXRα and PPARγ are determined. As the first report on the dual agonist targeting RXRα and PPARγ with receptor-ligand complex structures, our results are thus expected to help inspect the potential pharmacological mechanism for magnolol functions, and supply useful hits for nuclear receptor multi-target ligand design.

Hybrid-Based Multi-Target Ligands for the Treatment of Alzheimer's Disease

Progresses in medicinal chemistry over the last few years have focused on the design and synthesis of hybrid compounds, molecules encompassing in a single scaffold two pharmacophores from known entities endowed with well established biological activities. The interest in this topic is related to the increasing emphasis on the identification of the different factors involved in a number of disorders, such as the complex multifactorial Alzheimer's disease (AD), and hybrid- based strategy has become a focal point in this medicinal chemistry field since it could lead to derivatives with an improved biological profile. Using this strategy, acetylcholinesterase inhibitors (AChEIs) have been extensively coupled with properly selected bioactive molecules to obtain homo- and heterodimers endowed with increased potency together with supplementary actions. In the past decade the inhibition of the AChE induced aggregation of the -amyloid peptide into the senile plaques, which is a key event in the neurotoxic cascade of AD, has been considered a relevant approach leading to several dual binding site inhibitors, able to contact both the peripheral anionic site of AChE and the active site. In recent years, pioneering efforts have been performed to obtain novel AChEIs that, beyond the capability to inhibit AChE, were able to hit a number of specific AD targets. In particular, these compounds proved to possess antioxidant, anti-inflammatory, or neuroprotective activities, useful to block or revert the progression of the disease. This review summarizes the progresses that have been made in the design of hybrid molecules for the treatment of AD.

Exploiting the lipoic acid structure in the search for novel multitarget ligands against Alzheimer’s disease

Lipoic acid (LA) is a natural antioxidant. Its structure was previously combined with that of the acetylcholinesterase inhibitor tacrine to give lipocrine (1), a lead compound multitargeted against Alzheimer’s disease (AD). Herein, we further explore LA as a privileged structure for developing multimodal compounds to investigate AD. First, we studied the effect of LA chirality by evaluating the cholinesterase profile of 1’s enantiomers. Then, a new series of LA hybrids was designed and synthesized by combining racemic LA with motifs of other known anticholinesterase agents (rivastigmine and memoquin). This afforded 4, which represents a step forward in the search for balanced anticholinesterase and antioxidant capacities.