Vitamin B3-Based Biologically Active Compounds As Inhibitors of Human Cholinesterases
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International Journal of Molecular Sciences Article Vitamin B3-Based Biologically Active Compounds as Inhibitors of Human Cholinesterases Antonio Zandona 1 , Gabriela Lihtar 1, Nikola Marakovi´c 1, Katarina Miš 2, Valentina Buši´c 3, Dajana Gašo-Sokaˇc 3, Sergej Pirkmajer 2 and Maja Katalini´c 1,* 1 Institute for Medical Research and Occupational Health, Ksaverska c. 2, HR-10001 Zagreb, Croatia; [email protected] (A.Z.); [email protected] (G.L.); [email protected] (N.M.) 2 Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, SLO-1001 Ljubljana, Slovenia; [email protected] (K.M.); [email protected] (S.P.) 3 Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, Kuhaˇceva20, HR-31000 Osijek, Croatia; [email protected] (V.B.); [email protected] (D.G.-S.) * Correspondence: [email protected]; Tel.: +385-1-4682-551 Received: 22 October 2020; Accepted: 27 October 2020; Published: 29 October 2020 Abstract: We evaluated the potential of nine vitamin B3 scaffold-based derivatives as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors, as a starting point for the development of novel drugs for treating disorders with cholinergic neurotransmission-linked pathology. As the results indicate, all compounds reversibly inhibited both enzymes in the micromolar range pointing to the preference of AChE over BChE for binding the tested derivatives. Molecular docking studies revealed the importance of interactions with AChE active site residues Tyr337 and Tyr124, which dictated most of the observed differences. The most potent inhibitor of both enzymes with Ki of 4 µM for AChE and 8 µM for BChE was the nicotinamide derivative 1-(40-phenylphenacyl)-3-carbamoylpyridinium bromide. Such a result places it within the range of several currently studied novel cholinesterase inhibitors. Cytotoxicity profiling did not classify this compound as highly toxic, but the induced effects on cells should not be neglected in any future detailed studies and when considering this scaffold for drug development. Keywords: AChE; BChE; neurodegenerative; Alzheimer’s; nicotinamide; cytotoxicity 1. Introduction The therapeutic approach for treating various neurodegenerative disorders and disorders with cholinergic neurotransmission-linked pathology is currently focused mostly on alleviating their symptoms and progression. One such disease is Alzheimer’s disease (AD), which affects the elderly, and myasthenia gravis, which affects people of any age. AD is a severe degenerative brain disorder leading to dementia, memory loss and disorders of thinking, behaviour and personality [1,2]. The pathology of the disorder has been well researched and is characterised by the formation of cytotoxic plaques of misfolded amyloid β-peptides, hyperphosphorylation of τ-protein in the cells and by a loss of cholinergic neurotransmission [1,2]. Considering that there is no adequate treatment or drug that would act on the cause of the AD, the inhibition of human cholinesterases (acetylcholinesterase, AChE, EC 3.1.1.7; and butyrylcholinesterase, BChE, EC 3.1.1.8) emerged as an important therapeutic regime [3–8]. This is important especially since a diminished level of ACh in the brain is one of the characteristics of AD, so drugs designed as AChE inhibitors prevent ACh from fast degradation and enable longer-lasting neuro signals [9,10]. Even though BChE’s involvement in AD is still under investigation [11,12], it is also capable of hydrolysing ACh and thus diminish the effort of an AChE-acting drug [13–15]. Therefore, the use of nonselective cholinesterase inhibitors may be beneficial to AD patients as well [15,16]. Int. J. Mol. Sci. 2020, 21, 8088; doi:10.3390/ijms21218088 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 8088 2 of 19 In contrast to AD, myasthenia gravis is an autoimmune disease affecting the neuromuscular junction [17]. This disease’s main pathology is characterised by antibodies attacking acetylcholine receptors (AChR), muscle-specific kinase (MUSK) and low-density lipoprotein receptor-related protein 4 (LRP4) or other functionally related molecules in the postsynaptic membrane at the neuromuscular junction [17]. The most affected are the eyes, face and throat muscles and, in the later stage of disease progression, the neck and limb muscles [17,18]. As in the case of AD, no cure exists for myasthenia gravis, but treatment can help relieve signs and symptoms [19]. Again, drugs acting on ChE, that increase the amount of acetylcholine at neuromuscular endplates after motor nerve stimulation, alleviate muscle weakness in all myasthenia gravis patients [19]. Numerous structures have been investigated as scaffolds for potential cholinesterase inhibitors for the treatment of both AD [20–24] and myasthenia gravis [25,26]. To this day, only three AChE-acting drugs have been approved in treating AD symptoms: the benzylpiperidine donepezil [27,28], the tertiary alkaloid galantamine [29] and the carbamate rivastigmine [29,30]. Aminoacridine tacrine as an anti-AChE acting drug was withdrawn from the market due to hepatotoxicity [31,32]. Carbamate pyridostigmine is the preferred drug for treating myasthenia gravis, though several other carbamates are available at the market like neostigmine, and oxalamide ambenonium chloride [33]. Due to many side effects as well as a low number of novel candidates entering the late stage of drug-development pipeline, new compounds of better characteristics are constantly being sought [25,34–36]. A newly synthesized group of compounds based on vitamin B3, nicotinamide, was recently described as potentially biologically active against several targets [37,38]. Nicotinamide derivatives have broad pharmacological properties especially as inhibitors of many enzymes involved in the pathogenesis of numerous illnesses [39]. They are investigated as antioxidative [40], anticancer [37], anti-angiogenic [41] and antinociceptive agents [42], but also as drugs for treating patients with neurodegenerative diseases [43]. In that sense, we described nine vitamin B3 derivatives (Figure1) as inhibitors of human AChE and BChE. The synthesis of these compounds has been previously conducted [44,45]. Since these compounds have quaternary nitrogen present in their structure, we performed an in silico analysis of their basic physicochemical characteristics to estimate whether any would possess some characteristic advantageous for acting wider than at the peripheral nervous system (PNS). Furthermore, we performed an in vitro cytotoxicity screening on neural-like (SH-SY5Y) and kidney cells (HEK293) to evaluate their potential to be considered a starting point for novel anti-ChE drugs. Int. J. Mol. Sci. 2020, 21, 8088 3 of 19 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 19 FigureFigure 1.1. ChemicalChemical structuresstructures ofof testedtested vitaminvitamin B3-derivativesB3-derivatives 11--99 (nicotinamide(nicotinamide form)form) andand knownknown ChEChE actingacting drugsdrugs (reversible(reversible inhibitors).inhibitors). 2. Results 2. Results 2.1. In Silico Physicochemical Characteristics of Nicotinamide Derivatives 2.1. In Silico Physicochemical Characteristics of Nicotinamide Derivatives According to the in silico determined physicochemical properties, most of the tested nicotinamides possessAccording some characteristics to the in silico that could determined work in theirphysicochemical favour, like molecularproperties, weight most (M),of numberthe tested of hydrogennicotinamides bond possess donors andsome acceptors characteristics (HBD andthat HBA)could andwork number in their of favour, rotating like bonds molecular (RB) (Figure weight2). However,(M), number allcompounds of hydrogen have bond a negative donors logandP value,acceptors as expected (HBD and due HBA) to the and presence number of a permanentof rotating positivebonds (RB) charge. (Figure This 2). indicates However, a low all compounds lipophilicity have and aa lownegative potential logPto value, be passively as expected transported due to the to centralpresence nervous of a permanent system (CNS). positive Furthermore, charge. This compounds indicates a low5, 7 andlipophilicity9 also have and a a higherlow potential value of to the be topologicalpassively transported polar surface to area central (TPSA) nervous than recommendedsystem (CNS). by Furthermore, the reference compounds values set for 5, a 7 blood–brain and 9 also barrierhave a (BBB)higher acting value drugof the [46 topological]. According polar to insurface silico area calculations, (TPSA) than a moderate recommended possibility by ofthe a reference potential passivevalues set transport for a blood–brain through the BBBbarrier was (BBB) predicted acting only drug for [46]. compound According8 (Table to 1in). silico calculations, a moderate possibility of a potential passive transport through the BBB was predicted only for compoundTable 1. 8In (Table silico prediction1). of tested nicotinamide derivatives (1–9) passive transport to central nervous system (CNS) through the BBB, according to Discovery Studio Client’s ADMET Descriptors protocol. R denotes substituent and its position on the phenyl ring. Compound R logBB * Level ** − 1 4-H 0.885 3 − 2 4-F 0.821 3 − 3 4-Cl 0.679 3 − 4 4-Br 0.654 3 − 5 4-NO 1.595 3 2 − 6 4-CH 0.735 3 3 − 7 4-OCH 1.031 3 3 − 8 4-Ph 0.416 2 − 9 2-OCH 1.031 3 3 − * Very high penetrants logBB 0.7; High penetrants 0 logBB < 0.7; Medium penetrants 0.52 < logBB < 0; Low penetrants logBB 0.52. ** Very≥ high