We studied the inhibitory activity of methylene blue (MB) gamma-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 muM and exhibited low potencies against CaE (9.8-26% inhibition at 20 muM). 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.
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.
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 gamma-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 pi-pi 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.
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)2P(O)X, where R=alkyl, X=- SCH(Hal)COOEt (Hal=Cl, Br), -SCHCl2, -SCH2Br, -OCH(CF3)R(1) (R(1)=C6H5, CF3, 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.
A series of O-carbamoylated 1,1,1,3,3,3-hexafluoroisopropanols of general formula RNHC(O)OCH(CF3)2, where R = CH3, n-C3H7, tert-C4H9, cyclo-C6H11, C6H5-CH2, C6H5, 4-Cl-C6H4, 3-Cl-C6H4, 3,4-Cl2-C6H3, and naphthylen-2-yl were synthesized. The reaction kinetics of the synthesized carbamates with human erythrocyte acetylcholinesterase (EC 3.1.1.7), horse serum butyrylcholinesterase (EC 3.1.1.8), and porcine liver carboxylesterase (EC 3.1.1.1) were studied. It was shown that the synthesized carbamates did not inhibit acetylcholinesterase, inhibited weakly butyrylcholinesterase, and inhibited selectively the activity of carboxylesterase. A new selective irreversible inhibitor of carboxylesterase, 2,2,2-trifluoro-1-trifluoromethylethyl cyclohexylcarbamate, which had low acute toxicity, was obtained.
A modification of novel fluorinated organophosphorous compounds containing terminal alkyne group by different azidopeptides via Cu(I)-catalyzed click chemistry has been described. The inhibitor activity of trifluoromethyl-containing methylphosphonates and their peptide-conjugates towards acetylcholinesterase, butyrylcholinesterase, and carboxylesterase has been investigated. It was shown that the incorporation of peptide fragments significantly modulates the esterase profile of starting methylphosphonates.
This paper reviews previously published data and presents new results to address the hypothesis that fluorinated aminophosphonates (FAPs), (RO)(2)P(O)C(CF(3))(2)NHS(O)(2)C(6)H(5), R=alkyl, inhibit serine esterases by scission of the P-C bond. Kinetics studies demonstrated that FAPs are progressive irreversible inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7.), butyrylcholinesterase (BChE, EC 3.1.1.8.), carboxylesterase (CaE, EC 3.1.1.1.), and neuropathy target esterase (NTE, EC 3.1.1.5.), consistent with P-C bond breakage. Chemical reactivity experiments showed that diMe-FAP and diEt-FAP react with water to yield the corresponding dialkylphosphates and (CF(3))(2)CHNHS(O)(2)C(6)H(5), indicating lability of the P-C bond. X-ray crystallography of diEt-FAP revealed an elongated (and therefore weaker) P-C bond (1.8797 (13)A) compared to P-C bonds in dialkylphosphonates lacking alpha-CF(3) groups (1.805-1.822A). Semi-empirical and non-empirical molecular modeling of diEt-FAP and (EtO)(2)P(O)C(CH(3))(2)NHS(O)(2)C(6)H(5) (diEt-AP), which lacks CF(3) groups, indicated lengthening and destabilization of the P-C bond in diEt-FAP compared to diEt-AP. Active site peptide adducts formed by reacting diEt-FAP with BChE and diBu-FAP with NTE catalytic domain (NEST) were identified using peptide mass mapping with mass spectrometry (MS). Mass shifts (mean+/-SE, average mass) for peaks corresponding to active site peptides with diethylphosphoryl and monoethylphosphoryl adducts on BChE were 136.1+/-0.1 and 108.0+/-0.1Da, respectively. Corresponding mass shifts for dibutylphosphoryl and monobutylphosphoryl adducts on NEST were 191.8+/-0.2 and 135.5+/-0.1Da, respectively. Each of these values was statistically identical to the theoretical mass shift for each dialkylphosphoryl and monoalkylphosphoryl species. The MS results demonstrate that inhibition of BChE and NEST by FAPs yields dialkylphosphoryl and monoalkylphosphoryl adducts, consistent with phosphorylation via P-C bond cleavage and aging by net dealkylation. Taken together, predictions from enzyme kinetics, chemical reactivity, X-ray crystallography, and molecular modeling were confirmed by MS and support the hypothesis that FAPs inhibit serine esterases via scission of the P-C bond.
Title: Synthesis of organophosphates with fluorine-containing leaving groups as serine esterase inhibitors with potential for Alzheimer disease therapeutics Makhaeva GF, Aksinenko AY, Sokolov VB, Serebryakova OG, Richardson RJ Ref: Bioorganic & Medicinal Chemistry Lett, 19:5528, 2009 : PubMed
Acetylcholinesterase and butyrylcholinesterase inhibitors are potential cognition enhancers in Alzheimer disease. O,O-Dialkylphosphate inhibitors with 1-substituted 2,2,2-trifluoroethoxy leaving groups were synthesized by phosphonate-phosphate rearrangement. Substituents in the 1-position of the leaving group along with the O-alkyl groups modulated potency and selectivity against acetylcholinesterase, butyrylcholinesterase, and carboxylesterase.
Title: Study of the structural determinants of acute and delayed neurotoxicity of O-phosphorylated oximes by molecular field topology analysis (MFTA) Radchenko EV, Makhaeva GF, Sokolov VB, Palyulin VA, Zefirov NS Ref: Dokl Biochem Biophys, 429:309, 2009 : PubMed
Title: Modeling of the relationships between the structure of O-phosphorylated oximes and their anticholinesterase activity and selectivity using molecular field topology analysis (MFTA) Radchenko EV, Makhaeva GF, Malygin VV, Sokolov VB, Palyulin VA, Zefirov NS Ref: Dokl Biochem Biophys, 418:47, 2008 : PubMed
The title compound, C(13)H(16)F(6)NO(5)PS, is of inter-est with respect to inhibition of serine hydro-lases. Its structure contains a 1.8797 (13) A P-C bond and two inter-molecular N-Hcdots, three dots, centeredO=P hydrogen bonds, resulting in centrosymmetric dimers. An intra-molecular N-Hcdots, three dots, centeredO=P hydrogen bond is also present.
Title: Quantitative structure-activity relationships predict the delayed neurotoxicity potential of a series of O-alkyl-O-methylchloroformimino phenylphosphonates Malygin VV, Sokolov VB, Richardson RJ, Makhaeva GF Ref: J Toxicol Environ Health A, 66:611, 2003 : PubMed
Inhibition of acetylcholinesterase (AChE) versus inhibition and aging of neuropathy target esterase (NTE) by organophosphorus (OP) compounds in vivo can give rise to distinct neurological consequences: acute cholinergic toxicity versus OP compound-induced delayed neurotoxicity (OPIDN). Previous work has shown that the relative potency of an OP compound to react with NTE versus AChE in vitro may predict its capability to produce OPIDN. The present study was conducted to evaluate further the validity of such predictions and to enhance them with quantitative structure-activity relationships (QSAR) using a homologous series of alkyl phenylphosphonates (RO)C6H5P(O)ON = CCICH3 (PhP; R = alkyl). Neuropathic potential of PhP was assessed by measuring ki(NTE)ki(AChE) ratios in vitro and comparing these with ED50 ratios in vivo. Selectivity for NTE increased with rising R-group hydrophobicity. The ki(NTE)/ki(AChE) ratios were 0.42 (methyl), 3.6 (ethyl), 15 (isopropyl), 36 (propyl), 69 (isobutyl), 105 (butyl), and 124 (pentyl). Ratios > 1 suggest the potential to produce OPIDN at doses lower than the LD50. Inhibition of NTE and AChE in hen brain in vivo was studied 24 h after i.m. injection of hens with increasing doses of methyl and butyl derivatives. Analysis of dose-response curves yielded ED50(AChE)/ED50(NTE) ratio of 0.86 for methyl PhP and 22.1 for butyl PhP. These results predict that the butyl derivative should be more neuropathic than the methyl analogue. Excellent correspondence between in vivo and in vitro predictions of neuropathic potential indicate that valid predictive QSAR models may be based on the in vitro approach. Adoption of this system would result in reducing experimental animal use, lowering costs, accelerating data production, and enabling standardization of a biochemically based risk assessment of the neuropathic potential of OP compounds.
Title: [Paradoxical toxic effect and antagonism of O-(N-arylcarbamoylated) acylhydroxymoylchloride cholinesterase inhibitors with calcium]. [Russian] Ivanov I, Sokolov VB, Epishina TA, Martynov IV Ref: Doklady Akademii Nauk, 328:744, 1993 : PubMed
Title: Poster: The fluorocontaining derivatives of alpha-aminoalkylphosphonates a new type of cholinesterase inhibitors Kuusk VV, Agabekian RS, Morozova IV, Kovaleva NV, Rasdolsky AN, Sokolov VB, Aksinenko AY, Fetisov VI, Martynov IV Ref: In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology, (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC:293, 1991 : PubMed
Title: Poster: The structure-anticholinesterase activity relationships for new carbamoyloximes. QSAR study Shataeva GA, Sokolov VB, Fetisov VI, Ivanov YY, Epishina TA, Kovaleva NV, Martynov IV Ref: In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology, (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC:289, 1991 : PubMed
Title: [O-substituted alkylchloroformoximes as substrates and inhibitors of cholinesterases] Ivanov I, Sokolov VB, Epishina TA, Martynov IV Ref: Doklady Akademii Nauk SSSR, 310:1253, 1990 : PubMed
A series of O,O-diethyl-1-(N-alpha-hydrohexafluoroisobutyryl)aminoalkylphos phonates (APh) has been synthesized and their interaction with human erythrocyte acetylcholinesterase (AChE) and with horse serum butyrylcholinesterase (BuChE) studied. Most of the APhs inactivated the cholinesterases irreversible through formation of the enzyme-inhibitor intermediate. The inactivation rate constants and the enzyme-inhibitor intermediate dissociation constants are calculated. The quantitative structure-activity relationships including both hydrophobic and calculated steric parameters of substituents are developed for APh--ChE interactions. Molecular mechanics (programme MM2) was used for determining steric parameters (Es). On the basis of QSAR models analysis it was concluded that hydrophobic interactions play an essential role in APh--AChE binding, whereas for APh--BuChE binding steric interactions are essential. Presence of at least two APh binding centres on the surface of AChE and BuChE is suggested.
Title: [Comparative study of the interaction of acetylcholinesterases of human erythrocytes and the heads of houseflies with phosphorylated alkylchloroformoximes] Shataeva GA, Makhaeva GF, Iankovskaia VL, Sokolov VB, Ivanov AN Ref: Zhurnal Evoliutsionnoi Biokhimii i Fiziologii, 24:791, 1988 : PubMed
Studies have been made on the interaction of four types of phosphorylated alkylchloroformoximes, i.e. analogues of an insecticide-acaricide valexon, with acetylcholinesterases from human erythrocytes and from the heads of the housefly Musca domestica. Antiacetylcholinesterase activity of the drugs depended both on the structure of the organophosphorus compounds, and the origin of the enzyme, indicating the existence of differences in the active surface of these acetylcholinesterases. Incorporation of one or two chloride atoms into alkylchloroformoxime group of the cleaved part of the organophosphorus compounds increased anticholinesterase activity with respect to both enzymes. Diethyl derivatives of these drugs exhibited higher specificity with respect to housefly enzyme as compared to human acetylcholinesterase.
The interaction of dialkyl (alpha-carbometoxy-beta,beta,beta-trifluoroethyl) phosphates (RO)2P(O) . OCH(CF3)COOMe (R = Me, Et, Pr, Pri, Bu, Bui, Am, Hex) (I-VIII) with human erythrocyte acetylcholinesterase, horse serum butyrylcholinesterase, pig liver carboxylesterase was studied and acute toxicity in mice was estimated. Compounds (I)-(VIII) were not hydrolyzed by carboxylesterase, slowly and irreversibly inhibited acetylcholinesterase (kII = 10(2)-10(4) M-1 X min-1) and more efficiently inhibited butyrylcholinesterase and carboxylesterase (kII = 10(3)-10(7) M-1 X min-1). The structure--antienzymatic activity relationships were investigated. With increasing of hydrophobicity of alkoxy groups, antienzymatic activity to butyrylcholinesterase and carboxylesterase ("sites of loss") rises equally and more significantly, than antiacetylcholinesterase activity (delta lg kII 1.0 and 2.4 for R = CH3 and C5H11 resp.). Branching at the alpha-position of alkoxy groups leads to sharp reducing of acetylcholinesterase and butyrylcholinesterase inhibition constants, the carboxylesterase inhibition mechanism becoming reversible. Multiple regression analysis (the Kubinyi model) showed that influence of steric hindrances is revealed at the phosphorylation stage. It was found that phosphates (I)-(VIII) possess low acute toxicity in mice (900-2000 mg/kg). The toxicity of this homologous series appears to be independent of the hydrophobicity. Role of esterases in toxicological effect of compounds (I)-(VIII) is discussed.
