O. V. Dobrokhotova

Effect of Cyclic Hydroxamic Acids Derived from Glycine and D,L-Alanine on Activity of Ca2+, Mg2+-ATPase Hydrolases of Sarcoplasmic Reticulum and Cyclic Guanosine Monophosphate Phosphodiesterase

The effects of cyclic hydroxamic acids (CHAs) derived from glycine and D,L-alanine on the enzymatic activity of Ca2+,Mg2+-ATPase from sarcoplasmic reticulum (Ca2+,Mg2+-ATPase SR) and cyclic guanosine monophosphate phosphodiesterase (PDEcGMP) were investigated. CHAs I (C5H10N2O2), II (C6H12N2O2), III (C8H15N3O2), IV (C9H17N3O2), V (C11H21N3O2), and VI (C12H23N3O2) were modulators of Ca2+,Mg2+-ATPase SR enzyme activity. Compounds I-VI decoupled to various extents the hydrolytic and transport functions of Ca2+,Mg2+-ATPase SR, disrupting the ratio of intra- and extracellular Ca2+ ions. This affected the adhesion of metastatic cells to capillary endothelium. Compounds IV and VI had the highest metastasis inhibition indices (MII%) for B-16 melanoma of 33 and 81%, respectively. This correlated with a decreased Ca2+ transmembrane transfer coefficient into SR vesicles of 0.75 for IV and 0.5 for VI compared with a [Ca2+]/[ATP] ratio in the control of 1.4. CHAs I-VI did not affect the functioning of PDEcGMP. The results enabled potential antimetastatic drugs in the CHA series to be predicted.

Effect of nitrosyl iron–sulfur complexes on the activity of hydrolytic enzymes

We have studied the effect of some NO donors based on new nitrosyl iron—sulfur complexes, including the anionic thiosulfate complex Na2[Fe2(S2O3)2(NO)4](4H2O (I) and neutral thiolate complexes of the general formula [Fe2(SR)2(NO)2] with R = 1-methylimidazol-2-yl (II), 3-amino-1,2,4-triazol-5-yl (III), and phenyl (IV), on the enzymatic activity of two hydrolytic enzymes, namely, cyclic guanosine monophosphate phosphodiesterase (cGMP PDEase) and Ca2+—Mg2+-dependent ATPase of sarcoplasmatic reticulum (SR Ca2+—ATPase). It has been found that all tested compounds in the concentration range 0.1 – 0.001 mM inhibit functioning of cGMP PDEase and SR Ca2+—ATPase. Compounds I and II inhibit the activity of cGMP PDEase with Ki = 1 and 5 μM, respectively. These compounds are more efficient than the reference drug nicorandil (Ki = 0.1 mM) used in clinical therapy. The inhibition of cGMP PDEase functioning favors the accumulation of cGMP, a secondary messenger in living organisms that is responsible for vasodilatory, antiaggregant, and antihypertensive properties of drugs. Inhibition of SR Ca2+—ATPase blocks active transport of Ca2+ ions, thus affecting the equilibrium of Ca ions in cells and the manifestation of the above pharmacological effects. The experimental data allow one to predict the mechanisms of action of chemical compounds under consideration as potential drugs.

Action of Iron Nitrosyl Complexes, NO Donors, on the Activity of Sarcoplasmic Reticulum Ca2+-ATPase and Cyclic Guanosine Monophosphate Phosphodiesterase

The effect of iron nitrosyl complexes, NO donors, of a general formula [Fe2(L)2(NO)4] with functional sulfur-containing ligands (L-3-nitro-phenol-2-yl, 4-nitro-phenol-2-yl, or 1-methyl-tetrazol-5-yl) on the activity of sarcoplasmic reticulum Ca2+-ATPase and cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) was studied. The test complexes uncoupled the hydrolytic and transport functions of Ca2+- ATPase, thus disturbing the balance of Ca2+ ions in cells, which may affect the formation of thrombi and adhesion of metastatic cells to the endothelium of capillaries. They also inhibited the activity of cGMP PDE, thereby contributing to the accumulation of the second messenger cGMP. The studied iron nitrosyl complexes can be considered as potential drugs.

Effects of Nitrosyl Iron Complexes with Thiocarbamide and Its Aliphatic Derivatives on Activities of Ca 2+ -ATPase of Sarcoplasmic Reticulum and cGMP Phosphodiesterase

We studied the effects of water-soluble cationic dinitrosyl iron complexes with thiocarbamide and its aliphatic derivatives, new synthetic analogs of natural NO donors, active centers of nitrosyl [1Fe-2S]proteins, on activities of Ca2+-ATPase of sarcoplasmic reticulum and cGMP phosphodiesterase. Nitrosyl iron complexes [Fe(C3N2H8S)Cl(NO)2]0[Fe(NO)2(C3N2H8S)2]+Cl—(I), [Fe(SC(N(CH3)2)2(NO)2]Cl (II), [Fe(SC(NH2)2)2(NO)2Cl×H2O (III), and [Fe(SC(NH2)2)2(NO)2]2SO4×H2O (IV) in a concentration of 10—4 M completely inhibited the transporting and hydrolytic functions of Ca2+-ATPase. In a concentration of 10—5 M, they inhibited active Ca2+ transport by 57±6, 75±8, 80±8, and 85±9% and ATP hydrolysis by 0, 40±4, 48±5, and 38±4%, respectively. Complex II reversibly and noncompetitively inhibited the hydrolytic function of Ca2+-ATPase (Ki=1.7×10—6 M). All the studied iron—sulphur complexes in a concentration of 10—4 M inhibited cGMP phosphodiesterase function. These data suggest that the studied complexes can exhibit antimetastatic, antiaggregation, vasodilatatory, and antihypertensive activities.

Effects of Fullerene Derivatives on Activity of Ca2+-ATPase of the Sarcoplasmic Reticulum and cGMP Phosphodiesterase

We studied the effects of new water-soluble polysubstituted fullerene C60 (PFD) derivatives on activity of Ca2+-Mg2+ ATPase of the sarcoplasmic reticulum and cGMP phosphodiesterase. All examined fullerene derivatives inhibited activity of both enzymes. For instance, PFD-I, PFD-II, PFD-III, PFD-V, PFD-IX, PFD-X, and PFD-XI in a concentration of 5×10—5 M completely inhibited hydrolytic and transport functions of Ca2+-ATPase. These compounds in a concentration of 5×10—6 suppressed active transport of calcium ions by 51±5, 77±8, 52±5, 52±5, 100±10, 80±8, and 100±10%, respectively, and inhibited ATP hydrolysis by 31±3, 78±8, 18±2, 29±3, 78±8, 63±7, and 73±9%, respectively, uncoupling the hydrolytic and transport functions of the enzyme. PFD-I noncompetitive and reversibly reduced activity of Ca2+-ATPase (Ki=2.3×10—6 M). All the studied fullerene derivatives (except for PFD-VII) inhibited cGMP phosphodiesterase by more than 80% in concentration of 10—4 M and higher and by more than 50% in concentration of 10—5 M. PFD-I is a non-competitive reversible inhibitor of cGMP phosphodiesterase (Ki=7×10—6 M). These results allow us to expect antimetastatic, antiaggregatory, antihypertensive and vasodilative activity of the studied compounds.

Effect of Mexidol and Nitroxymexidol on Phosphodiesterase Activity, Some Oxidation Processes, and Hypoxia Resistance

The effect of mexidol (M) and nitroxymexidol (NM) on the activity of phosphodiesterase of cyclic guanosine monophosphate (PDEcGMP), regulation of lipid peroxidation (LPO), and antiradical and antihypoxic activity was investigated under normoxia and normobaric hypercapnic hypoxia conditions. Reversible and noncompetitive inhibition of the hydrolytic function of PDEcGMP by M and NM was revealed. The Ki value for M was 1 ×10–4 M; for NM, 1 ×10–5 M, i.e., NM was 10 times more active than M. It was shown that the lifetime of experimental animals in a closed space increased by 36% under the action of M as compared with that of the control group while it increased by 53% for NM. The ventricular contraction time increased by 137% for NM. The atrial contraction time increased from 31 min in the control to 52 and 57 min for M and NM, respectively.

Nicotinic acid nitroxyalkylamides as NA/K-ATPase inhibitors

The inhibitory action of nicotinamide N-nitroxyalkylamide derivatives (NANDs) on the hydrolase activity of Na/K-ATPase was studied. NANDs contain two peptide bonds and fragments of amides of glycine, β-alanine, α-aminopropanecarboxylic acid, or γ-aminobutyric acid. It is established that the inhibitory effect of NANDs depends on the concentration and hydrophobicity. An increase in the hydrophobicity of NANDs is accompanied by a decrease in their inhibitory action. The most active inhibitor among the studied NANDs is N-nitroxyethylnicotinamide, which noncompetitively and reversibly interacts with Na/K-ATPase with Ki = 2.13 × 10–4 M.

Influence of amitriptyline, chlorpromazine, and imipramine on the activity of phosphohydrolases

We have studied the effects of the antidepressants amitriptyline and imipramine and the neuroleptic chlorpromazine on the enzymatic function of Ca2+-activating-Mg2+-dependent ATPase of sarcoplasmic reticulum (SR), Na+, K+-dependent ATPase of endoplasmic reticulum (ER), and phosphodiesterase of cyclic adenosine monophosphate (cAMP). It is shown that interference with the enzymatic function (decrease of [Ca2+]/[ATP]) or inhibition of active transport of calcium ions by Ca2+, Mg2+-ATPase of SR by the psychotropic drugs leads to impairment of Ca2+ transport into the SR vesicule, which increases the force of muscle contraction. This effect decreases with increasing concentrations of amitriptyline and imipramine and with decreasing concentration of chlorpromazine, which is evidence for different mechanisms of action of Ca2+, Mg2+-ATPase of SR. Amitriptyline was a noncompetitive reversible inhibitor of Ca2+, Mg2+-ATPase of SR with an ATP hydrolysis inhibition constant Ki = 1.8 × 10−4 M and an active calcium transport inhibition constant Ki = 0.7 × 10−4 M. The goal of the investigation was to study the molecular mechanisms of action of amitriptyline, imipramine, and chlorpromazine.

Modulation of contractile enzyme activity by anticonvulsant drugs

Anticonvulsant drugs of the heterocyclic amide family inhibit in vitro enzymatic activity of Ca2+, Mg2+-ATPase of sarcoplasmic reticulum, the activated transmembrane calcium-ion transfer/ATP hydrolysis specific rate ratio being retained to provide lower concentrations of calcium ions in myofibrilla and relaxed muscles. Ethosuximide is a noncompetitive inhibitor with reversible action on basic enzyme catalyzed processes. The inhibition of the hydrolytic activity of the enzyme under the action of ethosuximide, phenytoin, and primidone was compared to their effect on other phosphatases (cAMP phosphodiesterase and Na+, K+-dependent ATPase).