Conjugates of Methylene Blue with Cycloalkaneindoles as New Multifunctional Agents for Potential Treatment of Neurodegenerative Disease.

The development of multi-target-directed ligands (MTDLs) would provide effective therapy of neurodegenerative diseases (ND) with complex and nonclear pathogenesis. A promising method to create such potential drugs is combining neuroactive pharmacophoric groups acting on different biotargets involved in the pathogenesis of ND. We developed a synthetic algorithm for the conjugation of indole derivatives and methylene blue (MB), which are pharmacophoric ligands that act on the key stages of pathogenesis. We synthesized hybrid structures and performed a comprehensive screening for a specific set of biotargets participating in the pathogenesis of ND (i.e., cholinesterases, NMDA receptor, mitochondria, and microtubules assembly). The results of the screening study enabled us to find two lead compounds (4h and 4i) which effectively inhibited cholinesterases and bound to the AChE PAS, possessed antioxidant activity, and stimulated the assembly of microtubules. One of them (4i) exhibited activity as a ligand for the ifenprodil-specific site of the NMDA receptor. In addition, this lead compound was able to bypass the inhibition of complex I and prevent calcium-induced mitochondrial depolarization, suggesting a neuroprotective property that was confirmed using a cellular calcium overload model of neurodegeneration. Thus, these new MB-cycloalkaneindole conjugates constitute a promising class of compounds for the development of multitarget neuroprotective drugs which simultaneously act on several targets, thereby providing cognitive stimulating, neuroprotective, and disease-modifying effects.

Conjugation of Aminoadamantane and γ-Carboline Pharmacophores Gives Rise to Unexpected Properties of Multifunctional Ligands.

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer's disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure-activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced ß-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.

Conjugates of methylene blue with γ-carboline derivatives as new multifunctional agents for the treatment of neurodegenerative diseases.

We studied the inhibitory activity of methylene blue (MB) γ-carbolines (gC) conjugates (MB-gCs) against human erythrocyte acetylcholinesterase (AChE), equine serum butyrylcholinesterase (BChE), and a structurally related enzyme, porcine liver carboxylesterase (CaE). In addition, we determined the ability of MB-gCs to bind to the peripheral anionic site (PAS) of Electrophorus electricus AChE (EeAChE) and competitively displace propidium iodide from this site. Moreover, we examined the ability of MB-gCs to scavenge free radicals as well as their influence on mitochondrial potential and iron-induced lipid peroxidation. We found that MB-gCs effectively inhibited AChE and BChE with IC50 values in the range 1.73-10.5 µM and exhibited low potencies against CaE (9.8-26% inhibition at 20 µM). Kinetic studies showed that MB-gCs were mixed-type reversible inhibitors of both cholinesterases. Molecular docking results showed that the MB-gCs could bind both to the catalytic active site and to the PAS of human AChE and BChE. Accordingly, MB-gCs effectively displaced propidium from the peripheral anionic site of EeAChE. In addition, MB-gCs were extremely active in both radical scavenging tests. Quantum mechanical DFT calculations suggested that free radical scavenging was likely mediated by the sulfur atom in the MB fragment. Furthermore, the MB-gCs, in like manner to MB, can restore mitochondrial membrane potential after depolarization with rotenone. Moreover, MB-gCs possess strong antioxidant properties, preventing iron-induced lipid peroxidation in mitochondria. Overall, the results indicate that MB-gCs are promising candidates for further optimization as multitarget therapeutic agents for neurodegenerative diseases.

Novel conjugates of aminoadamantanes with carbazole derivatives as potential multitarget agents for AD treatment.

A new group of compounds, promising for the design of original multitarget therapeutic agents for treating neurodegenerative diseases, based on conjugates of aminoadamantane and carbazole derivatives was synthesized and investigated. Compounds of these series were found to interact with a group of targets that play an important role in the development of this type of diseases. First of all, these compounds selectively inhibit butyrylcholinesterase, block NMDA receptors containing NR2B subunits while maintaining the properties of MK-801 binding site blockers, exert microtubules stabilizing properties, and possess the ability to protect nerve cells from death at the calcium overload conditions. The leading compound C-2h has been shown the most promising effects on all analyzed parameters. Thus, these compounds can be regarded as promising candidates for the design of multi-target disease-modifying drugs for treatment of AD and/or similar neuropathologies.

Esterase profiles of organophosphorus compounds in vitro predict their behavior in vivo.

We studied 4 serine esterases (EOHs) that are associated with the following consequences from their inhibition by organophosphorus compounds (OPCs): acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE; inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity, OPIDN). The relative degree of inhibition of these EOHs constitutes the "esterase profile" of an OPC, which we hypothesize can serve as a predictor of its overall physiological effects. To test this hypothesis, we selected 3 OPCs known from previous work on reference enzymes to span a wide range of esterase profiles, neuropathic potential, and acute cholinergic toxicity. For each compound, we determined in vitro IC50 and in vivo ED50 values for inhibition of AChE, BChE, CaE, and NTE in mouse brain and blood. The results showed good correlations between in vitro and in vivo measures of potency and selectivity except for brain CaE, a tissue-specific isoform of the enzyme that was less sensitive to the test compounds than expected. Thus, this synthesis of new and previously published results indicates that the concept of the esterase profile of OPCs is useful for the prediction of therapeutic and toxic effects in vivo.

Conjugates of γ-Carbolines and Phenothiazine as new selective inhibitors of butyrylcholinesterase and blockers of NMDA receptors for Alzheimer Disease.

Alzheimer disease is a multifactorial pathology and the development of new multitarget neuroprotective drugs is promising and attractive. We synthesized a group of original compounds, which combine in one molecule γ-carboline fragment of dimebon and phenothiazine core of methylene blue (MB) linked by 1-oxo- and 2-hydroxypropylene spacers. Inhibitory activity of the conjugates toward acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and structurally close to them carboxylesterase (CaE), as well their binding to NMDA-receptors were evaluated in vitro and in silico. These newly synthesized compounds showed significantly higher inhibitory activity toward BChE with IC50 values in submicromolar and micromolar range and exhibited selective inhibitory action against BChE over AChE and CaE. Kinetic studies for the 9 most active compounds indicated that majority of them were mixed-type BChE inhibitors. The main specific protein-ligand interaction is π-π stacking of phenothiazine ring with indole group of Trp82. These compounds emerge as promising safe multitarget ligands for the further development of a therapeutic approach against aging-related neurodegenerative disorders such as Alzheimer and/or other pathological conditions.

Organophosphorus compound esterase profiles as predictors of therapeutic and toxic effects.

Certain organophosphorus compounds (OPCs) inhibit various serine esterases (EOHs) via phosphorylation of their active site serines. We focused on 4 EOHs of particular toxicological interest: acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity, OPIDN). The relative degree of inhibition of these EOHs constitutes the "esterase profile" of an OPC and serves as a major determinant of its net physiological effects. Thus, understanding and controlling the esterase profile of OPC activity and selectivity toward these 4 target enzymes is a significant undertaking. In the present study, we analyzed the inhibitor properties of 52 OPCs against the 4 EOHs, along with pairwise and multitarget selectivities between them, using 2 QSAR approaches: Hansch modeling and Molecular Field Topology Analysis (MFTA). The general formula of the OPCs was (RO)(2)P(O)X, where R = alkyl, X = - SCH(Hal)COOEt (Hal = Cl, Br), -SCHCl(2), -SCH(2)Br, -OCH(CF(3))R(1) (R(1) = C(6)H(5), CF(3), COOEt, COOMe). The Hansch model showed that increasing neuropathic potential correlated with rising R hydrophobicity; moreover, OPC binding to scavenger EOHs (BChE and CaE) had different effects on potential acute and delayed neurotoxicity. Predicted protective roles of BChE and CaE against acute toxicity were enhanced with increasing hydrophobicity, but projected protection against OPIDN was decreased. Next, Molecular Field Topology Analysis (MFTA) models were built, considering atomic descriptors, e.g., effective charge, van der Waals radius of environment, and group lipophilicity. Activity/selectivity maps confirmed predictions from Hansch models and revealed other structural factors affecting activity and selectivity. Virtual screening based on multitarget selectivity MFTA models was used to design libraries of OPCs with favorable esterase profiles for potential application as selective inhibitors of CaE without untoward side effects.

Chronic administration of dimebon ameliorates pathology in TauP301S transgenic mice.

Dimebon belongs to a fast-growing group of "old" drugs that were suggested to be effective for therapy of pathological conditions different from their original targets. Following initial reports of successful Phase II clinical trials for mild-to-moderate Alzheimer's and Huntington's diseases, effects of Dimebon on various neurodegenerative conditions were investigated both in follow-up clinical trials and in various model systems. Although results of Phase III clinical trials carried out so far were disappointing, there is growing body of evidence that this drug can affect neuronal physiology in a way that would be beneficial at particular stages of development of certain types of neurodegeneration. To reveal what molecular and cellular pathological processes might be affected by Dimebon, we tested the ability of this drug to ameliorate pathology in model systems recapitulating particular pathogenic mechanisms involved in the development and progression of neurodegenerative diseases. Here we assessed the ability of Dimebon to modify several prominent features of tauopathies using transgenic tauP301S mice as a model. Chronic treatment with Dimebon was found to partially protect against the progressive decline in motor function and accumulation of tau-positive dystrophic neurons characteristic of tauP301S mice. Similar results were obtained with two further γ-carbolines structurally similar to Dimebon. Our data suggest that Dimebon and Dimebon-like compounds might be considered as drugs possessing disease-modifying activity for diseases with prominent tau pathology.