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.

Mitochondria as a promising target for developing novel agents for treating Alzheimer's disease.

The mitochondria-targeting drugs can be conventionally divided into the following groups: those compensating for the energy deficit involved in neurodegeneration, including stimulants of mitochondrial bioenergetics and activators of mitochondrial biogenesis; and neuroprotectors, that are compounds increasing the resistance of mitochondria to opening of mitochondrial permeability transition (MPT) pores. Although compensating for the energy deficit and inhibition of MPT are obvious targets for drugs used in the very early stages of Alzheimer-like pathology, but their use as the monotherapy for patients with severe symptoms is unlikely to be sufficiently effective. It would be optimal to combine targets that would provide the cognitive-stimulating, the neuroprotective effects and the ability to affect specific disease-forming mechanisms. In the design of such drugs, assessment of their potential mitochondrial-targeted effects is of particular importance. The possibility of targeted drug design for simultaneous action on mitochondrial and neurotransmitter's receptors targets is, in particularly, based on the known interplay of various cellular pathways and the presence of common structural components. Of particular interest is directed search for multitarget drugs that would act simultaneously on mitochondrial calcium-dependent functions, the targets (receptors, enzymes, etc.) facilitating neurotransmission, and the molecular targets related to the action of so-called disease-modifying factors, in particular, the formation and overcoming of the toxicity of ß-amyloid or hyperphosphorylated tau protein. The examples of such approaches realized on the level of preclinical and clinical trials are presented below.

Pharmacological Sequestration of Mitochondrial Calcium Uptake Protects Neurons Against Glutamate Excitotoxicity.

Neuronal excitotoxicity which is induced by exposure to excessive extracellular glutamate is shown to be involved in neuronal cell death in acute brain injury and a number of neurological diseases. High concentration of glutamate induces calcium deregulation which results in mitochondrial calcium overload and mitochondrial depolarization that triggers the mechanism of cell death. Inhibition of mitochondrial calcium uptake could be potentially neuroprotective but complete inhibition of mitochondrial calcium uniporter could result in the loss of some physiological processes linked to Ca2+ in mitochondria. Here, we found that a novel compound, TG-2112x, can inhibit only the lower concentrations mitochondrial calcium uptake (induced by 100 nM-5 µM) but not the uptake induced by higher concentrations of calcium (10 µM and higher). This effect was not associated with changes in mitochondrial membrane potential and cellular respiration. However, a pre-treatment of neurons with TG-2112x protected the neurons against calcium overload upon application of toxic concentrations of glutamate. Thus, sequestration of mitochondrial calcium uptake protected the neurons against glutamate-induced mitochondrial depolarization and cell death. In our hands, TG-2112x was also protective against ionomycin-induced cell death. Hence, low rate mitochondrial calcium uptake plays an underestimated role in mitochondrial function, and its inhibition could protect neurons against calcium overload and cell death in glutamate excitotoxicity.