Structure Modeling of the Norepinephrine Transporter

The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a major role in the treatment of many central and peripheral nervous system diseases. In this study, we focused on the spatial structure of a NET constructed by homology modeling on Drosophila melanogaster dopamine transporter templates. We further examined molecular construction of primary binding pocket (S1) together with secondary binding site (S2) and extracellular loop 4 (EL4). The next stage involved docking of transporter inhibitors: Reboxetine, duloxetine, desipramine, and other commonly used drugs. The procedure revealed the molecular orientation of residues and disclosed ones that are the most important for ligand binding: Phenylalanine F72, aspartic acid D75, tyrosine Y152, and phenylalanine F317. Aspartic acid D75 plays a key role in recognition of the basic amino group present in monoamine transporter inhibitors and substrates. The study also presents a comparison of hNET models with other related proteins, which could provide new insights into their interaction with therapeutics and aid future development of novel bioactive compounds

In vitro and in silico ADME-Tox profiling and safety significance of multifunctional monoamine oxidase inhibitors targeting neurodegenerative deseases

Herein we report in vitro metabolic stability in human liver microsomes (HLMs), interactions with cytochrome P450 isoenzymes (CYP3A4, CYP2D6, and CYP2C9), and cytotoxicity analyses on HEK-293, HepG2, Huh7, and WTIIB cell lines of our most recent multitarget directed ligands PF9601N, ASS234, and contilisant. Based on these results, we conclude that (1) PF9601N and contilisant are metabolically stable in the HLM assay, in contrast to the very unstable ASS234; (2) CYP3A4 activity was decreased by PF9601N at all the tested concentrations and by ASS234 and contilisant only at the highest concentration; CYP2D6 activity was reduced by ASS234 at 1, 10, and 25 μM and by PF9601N at 10 and 25 μM, whereas contilisant increased its activity at the same concentrations; CYP2C9 was inhibited by the three compounds; (3) contilisant did not affect cell viability in the widest range of concentrations: up to 10 μM on HEK-293 cells, up to 30 μM on Huh7 cells, up to 50 μM on HepG2 cells, and up to 30 or 100 μM on WTIIB cells. Based on these results, we selected contilisant as a metabolically stable and nontoxic lead compound for further studies in Alzheimer's disease therapy.This study received financial support from the National Science Centre Poland (Grant No. 2016/23/D/NZ7/01328). J.M.-C. thanks AEI (Government of Spain) for grants PDI- 2019-105813RB-C21 and SAF2015-65586-R. J.M.-C. and F.L.- M. thank UCJC (Grants UCJC 2020-33 UCJC 2020-03) for support

Synthesis of novel indole derivatives as multi-target-directed ligands with potential application in the treatment of Alzheimer's disease

Choroba Alzheimera jest postępującym zaburzeniem neurodegeneracyjnym prowadzącym do demencji. Ma bardzo złożoną patogenezę przez co może wymagać złożonego leczenia. Jedną z możliwości terapii jest zastosowanie ligandów wielofunkcyjnych, które jednocześnie oddziałują z co najmniej dwoma celami biologicznymi zaangażowanymi w rozwój choroby. W niniejszej pracy przedstawiono syntezę nowych ligandów wielocelowych, które mogłyby poprawić funkcje kognitywne równocześnie wpływając na procesy prowadzące do rozwoju choroby.W części teoretycznej pracy omówiono patofizjologię choroby Alzheimera, zwracając szczególną uwagę na rolę agregacji β-amyloidu w tym procesie oraz agonistów receptora 5-HT4 jako substancji go modyfikujących. Na przykładzie donekoprydu przedstawiona została również idea ligandów wielofunkcyjnych oraz ich potencjalne znaczenie w terapii choroby Alzheimera. W części doświadczalnej pracy opisano syntezę nowych pochodnych indolu, o potencjalnej aktywności hamującej acetylo- i/lub butyrylocholinoesterazę oraz blokujących receptory serotoninergiczne 5-HT6. Związki te zostały otrzymane w wyniku wieloetapowej syntezy chemicznej, której pierwszym etapem było otrzymanie pierwszorzędowych amin alifatycznych zawierających w swojej budowie układ: N-benzylo-1H-indolu, N-benzyloindoliny, lub benzyloindolin-2-onu. Otrzymane aminy w reakcji aminowania redukcyjnego z odpowiednimi aldehydami dały cztery serie związków finalnych. W sumie otrzymano dwanaście związków finalnych, których tożsamość została potwierdzona wynikami analizy LC-MS, 1H NMR oraz 13C NMR.Alzheimer’s disease is a progressive neurodegenerative disorder leading to dementia. Its pathogenesis is highly complex and it seems it might require complex treatment. One of the possibilities here is the use of multi-target-directed ligands, compounds that can simultaneously interact with two or more biological targets involved in the development of the disease. The thesis presents the synthesis of novel multi-target-directed ligands that may improve cognitive functions simultaneously affecting also the progress of Alzheimer’s disease. In the theoretical part of the thesis, the pathophysiology of Alzheimer’s disease was discussed with particular emphasis on the critical role of β-amyloid peptide aggregation and 5-HT4 receptor agonists as disease-modifying substances. On the example of donecopride an idea of the multi-target-directed ligands and their potential role in anti-AD therapy were also presented.In the experimental part, a synthesis of novel indole derivatives with potential acetyl- and/or butyrylcholinesterase and serotoninergic receptors 5-HT6 inhibitory activity was described. These compounds were obtained in multistep chemical synthesis wherein in the first step primary aliphatic amines containing N-benzyl-1H-indole, N-benzylindoline or benzylindolin-2-one moiety were synthesized. These amines were then used in a reaction of reductive amination with appropriate aldehydes and gave four series of the final compounds. Overall, twelve final compounds were synthesized and their identity was confirmed by the results of LC-MS, 1H NMR and 13C NMR analysis

Serotonin 5-HT6</DN receptor ligands and butyrylcholinesterase inhibitors displaying antioxidant activity - design, synthesis and biological evaluation of multifunctional agents against Alzheimers disease

Neurodegeneration leading to Alzheimer’s disease results from a complex interplay of a variety of processes including misfolding and aggregation of amyloid beta and tau proteins, neuroinflammation or oxidative stress. Therefore, to address more than one of these, drug discovery programmes focus on the development of multifunctional ligands, preferably with disease-modifying and symptoms-reducing potential. Following this idea, herein we present the design and synthesis of multifunctional ligands and biological evaluation of their 5-HT(6) receptor affinity (radioligand binding assay), cholinesterase inhibitory activity (spectroscopic Ellman’s assay), antioxidant activity (ABTS assay) and metal-chelating properties, as well as a preliminary ADMET properties evaluation. Based on the results we selected compound 14 as a well-balanced and potent 5-HT(6) receptor ligand (K(i) = 22 nM) and human BuChE inhibitor (IC(50) = 16 nM) with antioxidant potential expressed as a reduction of ABTS radicals by 35% (150 μM). The study also revealed additional metal-chelating properties of compounds 15 and 18. The presented compounds modulating Alzheimer’s disease-related processes might be further developed as multifunctional ligands against the disease

Discovery of 1-(phenylsulfonyl)-1H-indole-based multifunctional ligands targeting cholinesterases and 5-HT6 receptor with anti-aggregation properties against amyloid-beta and tau

Multifunctional ligands as an essential variant of polypharmacology are promising candidates for the treatment of multi-factorial diseases like Alzheimer's disease. Based on clinical evidence and following the paradigm of multifunctional ligands we have rationally designed and synthesized a series of compounds targeting processes involved in the development of the disease. The biological evaluation led to the discovery of two compounds with favorable pharmacological characteristics and ADMET profile. Compounds 17 and 35 are 5-HT6R antagonists (Ki = 13 nM and Ki = 15 nM respectively) and cholinesterase inhibitors with distinct mechanisms of enzyme inhibition. Compound 17, a tacrine derivative is a reversible inhibitor of acetyl- and butyrylcholinesterase (IC50 = 8 nM and IC50 = 24 nM respectively), while compound 35 with rivastigmine-derived phenyl N-ethyl-N-methylcarbamate fragment is a selective, pseudo-irreversible inhibitor of butyrylcholinesterase (IC50 = 455 nM). Both compounds inhibit aggregation of amyloid β in vitro (75% for compound 17 and 68% for 35 at 10 μM) moreover, compound 35 is a potent tau aggregation inhibitor in cellulo (79%). In ADMET in vitro studies both compounds showed acceptable metabolic stability on mouse liver microsomes (28% and 60% for compound 17 and 35 respectively), no or little effect on CYP3A4 and 2D6 up to a concentration of 10 μM and lack of toxicity on HepG2 cell line (IC50 values of 80 and 21 μM, for 17 and 35 respectively). Based on the pharmacological characteristics and favorable pharmacokinetic properties, we propose compounds 17 and 35 as an excellent starting point for further optimization and in-depth biological studies