Synthesis and antioxidative activity of metalloporphyrins bearing 2,6-di-tert-butylphenol pendants.

The novel metalloporphyrins (M=HH, Fe, Mn, Co, Cu, Zn) bearing 2,6-di-tert-butylphenol pendants as antioxidant substituents, and a long chain hydrocarbon palmitoyl group have been synthesized. The oxidation of compounds by PbO2 leads to the formation of the corresponding 2,6-di-tert-butylphenoxyl radicals studied by EPR. The activity of porphyrins in lipid peroxidation has been examined using (1) in vitro lipid peroxidation induced by tert-butylhydroperoxide in respiring rat liver mitochondria, (2) in vitro lipid peroxidation in liver homogenates of Wistar strain rats, and (3) a model process of peroxidation of (Z)-octadec-9-enic (oleic) acid as a structural fragment of lipids. The activity of these compounds depends dramatically on the nature of metal and might be changed from antioxidative (M=HH, Mn, Cu, Zn) to indifferent (M=Co), and to pro-oxidative one (M=Fe). The anti- or pro-oxidative action of these compounds may be derived from the concurrence between the involvement of 2,6-di-tert-butylphenol pendants acting as radical scavengers and redox active metal center promoting oxidation processes. The results of this study suggest that the polytopic compounds combining in one molecule 2,6-di-tert-butylphenol pendants, metalloporphyrin moiety, and a palmitoyl group, are membrane active compounds and might be studied in an effort to find novel pharmaceutical agents.

[Mitochondria as the target for neuroprotectors].

Contemporary methods of treatment of neurodegenerative diseases (NDs) are limited and mainly symptomatic. Despite difference in particular clinical manifestations, NDs have a number of common features, the main of which is death of certain neuron population. The authors suppose that mitochondria and the phenomenon of mitochondrial permeability transition (MPT), playing the key role in cell death, may be a perspective target for the search of drugs with a capability of delaying the neurological deficit associated with NDs. The authors have demonstrated that some neurotoxins which are widely used to model neurodegenerative conditions are able to potentiate or even induce MPT. The neuroprotective effect of widely used cognition-enhancing drugs, such as tacrin, memantine, dimebon, N-acethylserotonine, and extract of Gingko biloba), may also be a result of their interaction with mitochondria. Thus, the search of drugs capable of preventing or breaking the cascade of cell death as a result of MPT suppression and, at the same time, compensating for the impaired brain functions, is very topical.

Effect of beta-amyloid peptide fragment 25-35 on nonselective permeability of mitochondria.

Beta-amyloid peptide fragment 25-35 potentiated phosphate- and calcium-induced opening of mitochondrial channels and caused swelling of mitochondria (even without exogenous calcium and phosphate). These changes were accompanied by accumulation of lipid peroxidation products in mitochondria. Specific inhibitors of mitochondrial channels ADP and cyclosporine A prevented beta-amyloid peptide-induced swelling of mitochondria. Our findings suggest that potentiation of mitochondrial channel opening is an important component of the neurodegenerative effect of beta-amyloid.

Dimebon and tacrine inhibit neurotoxic action of beta-amyloid in culture and block L-type Ca(2+) channels.

Dimebon, a Russian-made drug, inhibited toxic effects of beta -amyloid on cultured neurons. Excessive accumulation of beta-amyloid in the brain is characteristic of Alzheimer dementias. Antialzheimer preparations tacrine and dimebon improve survival of cerebellar granule cells during long-term incubation with Abeta25-35, the neurotoxic fragment of beta-amyloid. Both preparations can block potential-dependent Ca(2+) entry into neurons by about 20%, which is explained by their selective action on L-type Ca(2+) channels. It was assumed that the neuroprotective effect of dimebon and tacrine against Abeta25-35 partially depends on inhibition of potential-dependent Ca(2+) entry.

[Inhibition of enzymatic activity of alpha-thrombin by low molecular weight synthetic inhibitor]. / Ingibirovanie fermentativnoi aktivnosti alpha-trombina nizkomolekuliarnym sinteticheskim ingibitorom.

The effect of the organophosphoric inhibitor, SA-152, on the fibrinogen-coagulating and TAME-esterase activity of bovine alpha-thrombin was studied. The irreversible inhibition constants (k11 = 1.1 x 10(4) M-1.min-1,Ki = 0.7 x 10(-4) M, k2 = 0.8 min-1 towards the coagulating activity and kII = 0.7 x 10(4) M-1.min-1, Ki = 0.3 x 10(-4) M, k2 = 0.2 min-1 towards the esterase activity) were determined. The SA-152 inactivated alpha-thrombin was dialyzed and incubated with 0.5 M and 2.5 M NaCl and 10 mM TAME. There was no reconstitution of activity of the SA-152 modified alpha-thrombin after dialysis and treatment with high concentrations of NaCl and TAME. Heparin interactions with the anion-binding site of the high molecular weight recognition center in the alpha-thrombin molecule did not significantly influence the values of the kinetic constants for the enzyme inhibition by SA-152. This finding is consistent with the hypothesis on the irreversible binding of SA-152 in the active center of the enzyme.

[Regulation of thrombocyte stimulating and other activities of thrombin by modulators of the recognition site]. / Reguliatsiia trombotsitstimuliruiushchei i drugikh aktivnosti moduliatorami tsentra uznavaniia]

The structural and functional features of thrombin are under discussion: combination of restricted specificity and a central regulatory role in hemostasis. Thrombin specificity is mainly connected with special regions of the enzyme molecule--an additional recognition binding site for high molecular substrates. One can consider the additional site of thrombin as a kind of the allosteric centre changing thrombin-catalyzed functions at binding with modulator. Specific site of substrate (inhibitor or receptor) is used in the role of modulator. A computer search of that modulator was fulfilled by means of the program DOTHELIX. The peptides thymosin I and substance P which have regions similar to those of hirudin were shown to inhibit thrombin activity. The kinetic data point to the noncompetitive type of inhibition. The data on the high reactivity of the thrombin-activated protein C system confirm the idea of protein C to be the first defensive mechanism when thrombin is generated in blood and interacts with thrombomodulin.

[Effect of thrombomodulin on specific thrombin functions: fibrinogen coagulation and thrombocyte aggregation]. / Vliianie trombomodulina na spetsificheskie funktsii trombina: svertyvanie fibrinogena i agregatsiiu trombotsitov.

The effect of thrombomodulin (TM), prepared from rabbit lungs, on fibrinogen clotting and platelets aggregation by alpha-thrombin has been investigated. It has been established that TM caused a dose-dependent decrease in fibrinogen-clotting activity of thrombin (Ki = 14.7 +/- 1.24 nM). TM was shown to reduce thrombin-induced platelet aggregation but not to alter ADP-induced one. It was found that the kinetic parameters for hydrolysis of synthetic substrates by alpha-thrombin were not altered by TM.

[Modulation of the platelet aggregation capacity by modified forms of thrombin]. / Moduliatsiia modifitsirovannymi formami trombina aggregatsionnoi sposobnosti trombotsitov.

It was found that human platelets possess a high sensitivity towards alpha-thrombin (Km = 2 nM). Modified thrombin forms (beta/gamma-thrombin) with an impaired recognition site of high molecular weight substrates and DIP-alpha-thrombin and trypsin are incapable of inducing platelet aggregation when taken at concentrations corresponding to effective concentrations of alpha-thrombin. Beta/gamma-Thrombin and trypsin, unlike DIP-alpha-thrombin, cause platelet aggregation at concentrations of 100-200 nM. Studies on the modulating effects of modified thrombin forms, alpha-thrombin and trypsin, on platelet aggregation induced by alpha-thrombin revealed that beta/gamma-thrombin, alpha-thrombin and trypsin at concentrations causing no cell aggregation potentiate the platelet response after 2 min incubation and inhibit platelet aggregation upon prolonged (15 min) incubation. However, DIP-alpha-thrombin, irrespective of the incubation time (up to 30 min) increased the sensitivity of platelets to alpha-thrombin-induced aggregation. The activating effect of DIP-alpha-thrombin is characterized by an equilibrium constant (KA) of 17 nM. The experimental data confirm the hypothesis that the necessary prerequisite for an adequate physiological response of platelets to alpha-thrombin is the maintenance in the thrombin molecule of an intact active center and a recognition site for high molecular weight substrates. The specificity of thrombin as a potent platelet aggregation inducer is determined by the recognition site for high molecular weight substrates.

[Effect of prethrombin I and prostaglandin E2 on thrombin-stimulated aggregation of rat platelets]. / Vliianie pretrombina I i prostaglandina E2 na stimuliruemuiu trombinom agregatsiiu trombotsitov krys.

Effect of inactive precursor of alpha-thrombin--prethrombin (Pre-I) and prostaglandin (PGE2) on aggregation of washed rat platelets induced by alpha-thrombin was studied. Pre-I inhibited dose-dependently the alpha-thrombin-induced platelet aggregation. K1 values for Pre-I of high and low affinity binding sites were equal to 0.9.10(-8) M and 0.56.10(-6) M, respectively. PGE2 inhibited the alpha-thrombin-induced aggregation after simultaneous addition and within 5 and 15 min of incubation with PGE2. Preincubation of platelets with alpha-thrombin within 15 min prevented the PGE2-induced aggregation but preincubation of platelets with Pre-I within 15 min did not influence the platelet aggregation, induced by PGE2.

[Kininogenase activity of alpha and beta/gamma forms of bovine thrombin]. / Kininogenaznaia aktivnost' al'fa- i beta/gamma-form trombina byka.

The kininogenase activity of alpha- and beta/gamma-forms of bovine thrombin with respect to the high molecular weight (HMW) and low molecular weight (LMW) human kininogens was studied. It was shown that both forms of the enzyme split of bradykinin from these kininogens. The kininogenase activity of alpha-thrombin is completely blocked by the highly specific thrombin inhibitor Nalpha-dansyl-L-arginine-p-ethylpiperidineamide, but not by the soya bean trypsin inhibitor. The alpha- and beta/gamma-forms of thrombin hydrolyze HMW (Km(app) = 4.5 and 3.3 microM, respectively) and LMW (Km(app) = 10.1 and 4.7 microM, respectively). The specific constants (kcat/Km(app) ) for thrombin with respect to the substrates differ about 7-fold, predominantly due to the high catalytic rates of HMW as compared to LMW; the kcat values are 0.18 and 0.06 min-1, respectively. alpha-Thrombin upon a long-term (over 1 hour) exposure to HMW, besides bradykinin, splits off the product inhibiting the kininogenase activity of thrombin. No differences in the specificity of the beta/gamma-form of thrombin with resect to HMW and LMW were detected.

[Hydrolysis of methyl esters of N alpha-arylsulfonyl-arginine and N-arylsulfonyl-valyl-arginine by alpha- and beta/gamma-thrombins]. / Gidroliz metilovykh éfirov N. al'fa-arilsul'fonil-arginina i N-arilsul'fonil-valil-arginina al'fa i beta/gamma-trombinami.

The kinetic parameters of hydrolysis of methyl esters of N alpha-arylsulfonyl-arginine and N-arylsulfonyl-valyl-arginine by alpha- and beta/gamma-thrombins were calculated. It was found that the polarity and volume of arylsulfonyl substitute are essential for hydrolysis of N alpha-arylsulfonyl-arginine esters and only slightly affect the hydrolysis of N-arylsulfonyl-valyl-arginine esters. Incorporation of the valine residue into the substrate molecule sharply increases the catalytic constant, thus suggesting an important role of secondary sites of the enzyme binding to the substrate. A comparison of alpha- and beta/gamma-thrombins did not reveal any substantial differences in their ability to hydrolyze synthetic esters; consequently the structure of the region around the enzyme active center which is involved in interactions with the substrates under study, is not responsible for alpha-thrombin specificity for fibrinogen.

[Regulation of alpha and beta/gamma-thrombin activity by heparin and indole]. / Reguliatsiia aktivnosti alfa-i beta/gamma-form trombina geparinom i indolom.

Indole was shown to stimulate TAME hydrolysis by alpha-thrombin with KA=7,7 mM, alpha=0,55, beta=1,5 and that by beta/gamma-thrombin with KA=9,5 mM, alpha=0,47 and beta=2,08. Indole promotes both the formation and transformation of the enzyme-substrate complex. No effect of substrate activation was observed in the presence of indole, which suggests the identity of the binding sites of indole and the added TAME molecule. Heparin was shown to form an equimolar stoicheometric complex with both alpha- and beta/gamma-thrombin, which results in 40% inhibition of the TAME-esterase, clotting and prothrombinolytic activities of alpha-thrombin and 25% and 40% inhibition of the TAME-esterase and prothrombinolytic activity of beta/gamma-thrombin, respectively. The fact that alpha- and beta/gamma-thrombin partially retain their catalytic properties even at a 5-fold molar excess of the inhibitor indicates that heparin binds to the thrombin molecule at a site other than the active center. Heparin does not prevent the effect of substrate activation at high TAME concentrations. This finding suggests that the localization of binding sites of heparin and the added TAME molecule (and, therefore, indole) in the thrombin molecule is different.

[Enzymatic hydrolysis and reversible binding of suberyldicholine by cholinesterases]. / Fermentativnyi gidroliz i obratnoe sviazyvanie suberildikholina kholinesterazami

A hydrolysis of suberyldicholine and monocholic ester of suberic acid by butyrylcholinesterase was studied. In the region of Sopt the rates of suberyldicholine hydrolysis were slightly below and under S less than Sopt they were far in excess of the rates of acetylcholine hydrolysis. The following kinetic constants of hydrolysis were obtained: for suberyldicholine--Km=2.3-10(-5)M, V=2.4 mcM/mg. min, Kss=7.2-10(-2)M; for monocholic ester of suberic acid--Km=7.5-10(-4)M, V=1.5 mcM/mg, min, Kss=1.2-10(-2)M (25 degrees, pH 7.5, 0.1 M KCl). Suberyldicholine was shown to be highly active reversible inhibitor of competitive--non-competitive type (Ki=2.3-10(-6)M, alpha=0.5) of acetylcholinesterase from human erythrocytes; the inhibitory effect of monocholic ester of suberic acid was distinctly lower. By biological and indirect biochemical methods it was found that low concentrations of suberyldicholine 10(-5)=10(-6)M (similar to concentrations that were in an organism upon myorelaxation) were hydrolyzed by acetlycholinesterase with the rate, approximately equal to the rate of acetylcholine hydrolysis. The reversible binding and the enzymatic hydrolysis of suberyldicholine by acetylcholinesterase of tissues were likely to be the main factors that determined the effectiveness and prolonged blocking action of suberyldicholine on the nerve-muscle conductivity.