Ljuba M. Mandić

4-Aryl-4-oxo-N-phenyl-2-aminylbutyramides as acetyl- and butyrylcholinesterase inhibitors. Preparation, anticholinesterase activity, docking study, and 3D structure-activity relationship based on molecular interaction fields

Synthesis and anticholinesterase activity of 4-aryl-4-oxo-N-phenyl-2-aminylbutyramides, novel class of reversible, moderately potent cholinesterase inhibitors, are reported. Simple substituent variation on aroyl moiety changes anti-AChE activity for two orders of magnitude; also substitution and type of hetero(ali)cycle in position 2 of butanoic moiety govern AChE/BChE selectivity. The most potent compounds showed mixed-type inhibition, indicating their binding to free enzyme and enzyme-substrate complex. Alignment-independent 3D QSAR study on reported compounds, and compounds having similar potencies obtained from the literature, confirmed that alkyl substitution on aroyl moiety of molecules is requisite for inhibition activity. The presence of hydrophobic moiety at close distance from hydrogen bond acceptor has favorable influence on inhibition potency. Docking studies show that compounds probably bind in the middle of the AChE active site gorge, but are buried deeper inside BChE active site gorge, as a consequence of larger BChE gorge void.Synthesis and anticholinesterase activity of 4-aryl-4-oxo-N-phenyl-2-aminylbutyramides, novel class of reversible, moderately potent cholinesterase inhibitors, are reported. Simple substituent variation on aroyl moiety changes anti-AChE activity for two orders of magnitude; also substitution and type of hetero(ali)cycle in position 2 of butanoic moiety govern AChE/BChE selectivity. The most potent compounds showed mixed-type inhibition, indicating their binding to free enzyme and enzyme–substrate complex. Alignment-independent 3D QSAR study on reported compounds, and compounds having similar potencies obtained from the literature, confirmed that alkyl substitution on aroyl moiety of molecules is requisite for inhibition activity. The presence of hydrophobic moiety at close distance from hydrogen bond acceptor has favorable influence on inhibition potency. Docking studies show that compounds probably bind in the middle of the AChE active site gorge, but are buried deeper inside BChE active site gorge, as a consequence of larger BChE gorge void.

Influence of the microenvironment of thiol groups in low molecular mass thiols and serum albumin on the reaction with methylglyoxal.

Methylglyoxal (MG), a reactive alpha-oxoaldehyde that is produced in higher quantities in diabetes, uremia, oxidative stress, aging and inflammation, reacts with the thiol groups (in addition to the amino and guanidino groups) of proteins. This causes protein modification, formation of advanced glycated end products (AGEs) and cross-linking. Low molecular mass thiols can be used as competitive targets for MG, preventing the reactions mentioned above. Therefore, this paper investigated how the microenvironment of the thiol group in low molecular mass thiols (cysteine, N-acetylcysteine (NAcCys), carboxymethylcysteine (CMC) and glutathione (GSH)) and human serum albumin (HSA) affected the thiol reaction with MG. The SH group reaction course was monitored by (1)H-NMR spectroscopy and spectrophotometric quantification. Changes in the HSA molecules were monitored by SDS-PAGE. The microenvironment of the SH group had a major effect on its reactivity and on the product yield. The reactivity of SH groups decreased in the order Cys>GSH>NAcCys. CMC did not react. The percentages of the reacted SH groups in the equilibrium state were almost equal, regardless of the ratio of thiol compound/MG (1:1, 1:2, 1:5): 38.1 + or - 0.9%; 38.2 + or - 0.7% and 39.0 + or - 0.8% for Cys; 26.5 + or - 0.6%; 26.6 + or - 2.6% and 27.4 + or - 2.5% for GSH; 10.8 + or - 0.9%; and 11.2 + or - 0.7% and 12.2 + or - 0.9% for NAcCys, respectively. Our results explain why substances containing alpha-amino-beta-mercapto-ethane as a pharmacophore are successful scavengers of MG. In equilibrium, HSA SH reacted in high percentages both with an insufficient amount and with an excess of MG (55% and 65%, respectively). An analysis of the hydrophobicity of the microenvironment of the SH group on the HSA surface showed that it could contribute to high levels of SH modification, leading to an increase in the scavenging activity of the albumin thiol.

Fish oil supplementation improved liver phospholipids fatty acid composition and parameters of oxidative stress in male Wistar rats.

In the present study, we examined the effects of fish oil supplementation in 3 months old male Wistar rats on changes in plasma and liver lipid metabolism and oxidative stress parameters. Twenty Wistar rats were randomly divided into two groups of ten animals: control group and intervention group, treated for 6 weeks with fish oil capsules containing 45 mg eicosapentanoic acid and 30 mg docosahexanoic acid. After intervention, biochemical parameters in plasma [triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and total cholesterol, urea, creatinine and uric acid], fatty acid (FAs) profile of liver phospholipids and parameters of oxidative stress in liver [activity of catalase, superoxide dismutase and paraoxonase (PON1), concentration of nitrites, lipid peroxidation (LPO), free thiol (SH) groups and lactate dehydrogenase (LDH) izoenzymes were determined. Treatment with fish oil improved FAs profile of liver phospholipids, increasing n-3 FAs and decreasing n-6/n-3 ratio. Significant decrease in plasma TG and LDL concentration, and increase in the level of HDL and uric acid were found in intervention group at the end of the study. Catalase activity, LPO, and nitrites concentration in liver were significantly decreased, after the supplementation, together with elevated PON1 activity. Applied treatment significantly improved plasma lipid profile, liver FAs composition and parameters of oxidative stress in male Wistar rats.

Method for monitoring of the protein amino group changes during carbonylation

OBJECTIVES Carbonylation of the protein amino, guanidino and thiol groups is one of the important causes of vascular complications in diabetes. We developed a simple spectrophotometric method for monitoring of the changes in the protein amino group contents during carbonylation. DESIGN AND METHODS The method is based on the reaction of amino group with p-benzoquinone in the slightly alkaline media. It was applied during carbonylation in vitro with methylglyoxal and in vivo in 13 patients with type 2 diabetes and 20 healthy persons. RESULTS The method is simple, fast, precise (RSD in the range of 1.2-1.8%) and accurate (recovery about 100%). The content of amino groups in human serum albumin isolated from diabetics was significantly lower (p<0.01) in comparison with a control group. CONCLUSION The method developed is suitable for quantification of protein amino groups during in vitro carbonylation as well as for clinical practice.

Structural modifications of 4-aryl-4-oxo-2-aminylbutanamides and their acetyl- and butyrylcholinesterase inhibitory activity. Investigation of AChE–ligand interactions by docking calculations and molecular dynamics simulations

Congeneric set of thirty-eight 4-aryl-4-oxo-2-(N-aryl/cycloalkyl)butanamides has been designed, synthesized and evaluated for acetyl- and butyrylcholinesterase inhibitory activity. Structural variations included cycloalkylamino group attached to C2 position of butanoyl moiety, and variation of amido moiety of molecules. Twelve compounds, mostly piperidino and imidazolo derivatives, inhibited AChE in low micromolar range, and were inactive toward BChE. Several N-methylpiperazino derivatives showed inhibition of BChE in low micromolar or submicromolar concentrations, and were inactive toward AChE. Therefore, the nature of the cycloalkylamino moiety governs the AChE/BChE selectivity profile of compounds. The most active AChE inhibitor showed mixed-type inhibition modality, indicating its binding to free enzyme and to enzyme-substrate complex. Thorough docking calculations of the seven most potent AChE inhibitors from the set, showed that the hydrogen bond can be formed between amide -NH- moiety of compounds and -OH group of Tyr 124. The 10 ns unconstrained molecular dynamic simulation of the AChE-compound 18 complex shows that this interaction is the most persistent. This is, probably, the major anchoring point for the binding.

The influence of fatty acids on determination of human serum albumin thiol group.

During investigation of the changes of the Cys34 thiol group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11-33%) than the total serum thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8×10(-3), 21.6×10(-3), and 11.2×10(-3) s(-1), respectively. The HSA-SH group of diabetics is more reactive compared with control individuals (rate constants 20.9×10(-3)±4.4×10(-3) vs 12.9×10(-3)±2.6×10(-3) s(-1), P<0.05). Recovery values of the SH group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum thiols, which make the determination of the thiol group unreliable.

Monitoring of the human serum albumin carbonylation level through determination of guanidino group content

Carbonylation of the protein amino, guanidine, and thiol groups with α-oxoaldehydes (which are produced in higher quantities in diabetes, uremia, oxidative stress, aging, and inflammation) is one of the important causes of vascular complications. For monitoring of the human serum albumin (HSA) carbonylation level, a spectrophotometric method based on the formation of colored adduct between guanidine group and thymol-sodium hypobromite reagent in the alkaline medium was investigated. Beers law is obeyed in the concentration range of Arg and protein guanidine groups from 1 to 40 mM. Precision of the method (relative standard deviation) was in the range of 0.9 to 2%. Accuracy was examined by the standard addition method (recovery ~100%). The method was applied for monitoring of the carbonylation level of HSA with methylglyoxal in vitro and of HSA isolated (using affinity chromatography) from sera of 21 patients with type 2 diabetes and 12 healthy persons. The content of guanidine groups in HSA isolated from diabetics (19.64 ± 1.07 mM/mM albumin) was significantly lower (P < 0.001) in comparison with a control group (21.87 ± 1.02 mM/mM albumin). The method is simple and fast, has good accuracy and precision, and is suitable for clinical practice as well for in vitro protein carbonylation experiments.

Changes of isoenzymes of serum N‐acetyl‐β‐D‐glucosaminidase in relation to different types of diabetes

The activity of the isoenzymes of N‐acetyl‐β‐D‐glucosaminidase (NAG, EC 3.2.1.30) is determined in the serum of insulin‐dependent (IDDM) and non‐insulin‐dependent diabetics (NIDDM) with or without diabetic complications. The increase in total activity of serum NAG in diabetics is proportional to the A form activity (r=0,976, p< 0,0001). The contribution of the A form activity (65.87±5.99%) to total NAG activity of IDDM and NIDDM diabetics with and without complications does not change considerably compared to the control group. The contribution of the B form activity depends on the state of metabolic monitoring and diabetic complications. A significantly lower activity of the serum B form was found in IDDM (p<0,001) and NIDDM diabetics (p<0,05) compared to the healthy individuals, as well as the higher activity ratios of the A/B forms. A decrease in serum B form is correlated with the occurrence and abundance of the intermediary I forms (r=‐0,665). These changes are particularly significant in the individuals with the pronounced microangiopathy.

Quantification of total content of non-esterified fatty acids bound to human serum albumin

Non-esterified fatty acids bound to the human serum albumin (HSA) contribute to several HSAs properties of special concern in pathologies, for instance to the reactivity of the free HSA-Cys34 thiol group (important antioxidative thiol pool in plasma), and to the affinity for binding of molecules and ions (for example cobalt as a prominent biomarker in heart ischemia). Therefore, the method for determination of FAs bound to HSA was developed. FAs were released from HSA (previously isolated from serum by ammonium sulfate precipitation) using acidic copper(II) sulfate in phosphoric acid, extracted by n-heptane-chloroform (4:1, v/v) mixture, spotted on TL silica-gel and then developed with n-heptane-chloroform-acetic acid (5:3:0.3, v/v/v). Common office flatbed scanner and software solution for densitometric image analysis, developed in R, were used. The linearity of calibration curve in concentration range from 0.1 to 5.0mmol/L stearic acid was achieved. The method was proved to be precise (with RSD of 1.4-4.7%) and accurate. Accuracy was examined by standard addition method (recoveries 97.2-102.5%) and by comparison to results of GC. The method is sample saving, technically less demanding, and cheap, and therefore suitable for determination of FAs/HSA ratio when elevated concentrations of free FAs are reliable diagnostic/risk parameter of pathological states.

Serum N‐acetyl‐β‐D‐glucosaminidase profiles in type 1 diabetes secondary complications: causes of changes and significance of determination

The connection between changes in the activity of serum N‐acetyl‐β‐D‐glucosaminidase (NAG, E.C.3.2.1.30) and iso‐enzymes and degree of secondary complications was analyzed in four groups of type 1 diabetic patients (n=69): without complications (n=22); with retinopathy (n=16); with retinopathy and polyneuropathy (n=13), and with retinopathy, neuropathy, and nephropathy (n=18). In all groups statistically significant higher (P<0.001) percent fraction of A form (83.84±6.09, 84.37±5.74, 81.76±6.02, 76.37±7.38%, resp.) and lower (P<0.001, P<0.01) fraction of B form (15.87±5.65, 15.66±5.74, 18.33±5.98, 23.63±7.38, resp.) in total NAG compared with the control (A=69.38±4.79%, B=30.61±4.78%) were found. The differences in A as well as B forms between diabetic groups were not statistically significant. Significant strong positive correlations between total NAG and glycemia (0.494–0.623), total NAG and A form (0.934–0.966), and A form and glycemia (0.512–0.638) were found in all groups. No correlation was found between the fractions of B and A forms, except in the fourth group. The A form of diabetic patients in the fourth group was more acidic compared with the control and other diabetic groups. It was concluded that the changes in serum NAG and iso‐enzymic profiles in diabetes are the consequence of its increased exocytose, especially of the A form, in hyperglycemia and posttranslational modifications of iso‐enzymes. The total activity of serum NAG and iso‐enzymic profiles cannot be used for monitoring the development and distinction of type 1 diabetes secondary complications. J. Clin. Lab. Anal. 22:307–313, 2008.