Tag Archives: inhibitors

1,3-Oxazol-4-ylphosphonium salts as new non-peptide inhibitors of furin

T. V. Osadchuk1, V. K. Kibirev1,2, O. V. Shybyryn1, A. V. Semyroz1,
Ye. S. Velihina1, Е. R. Abdurakhmanova1, V. S. Brovarets1

1V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry,
National Academy of Sciences of Ukraine, Kyiv;
e-mail: brovarets@bpci.kiev.ua;
2Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv

Received: 22 February 2019; Accepted: 17 May 2019

A series of novel triphenylphosphonium derivatives of 1,3-oxazole containing at C2 and C5-positions electron withdrawing or electron-donating groups were synthesized and characterized by 1H, 31P NMR and IR spectroscopy, element analysis and chromato-mass spectrometry. These compounds were found to be a new class of non-peptide inhibitors of furin. Depending on the chemical structure, they inactivated enzyme at micromolar level by mechanism of competitive, non-competitive or mixed inhibition. Evaluation of the synthesized derivatives as furin inhibitors showed that among the triphenylphosphonium salts studied by us, oxazole 12 containing 2,4-dichlorophenyl- in the C2-position and MeS-group at C5 is the most active (Ki = 1.57 μM) competitive inhibitor of furin. Our results provided evidence that chemical modification of 1,3-oxazole-4-yl-triphenylphosphonium salts may be useful for developing new more potent and selective inhibitors of furin.

Role of charge and hydrophobic effects in reactions of peptide substrates and inhibitors with thrombin

A. A. Poyarkov, V. V. Prokopenko, S. A. Poyarkova

Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: alexp@bpci.kiev.ua

A substrate and inhibitor analysis of the thrombin interaction with synthetic peptide substrates and inhibitors of differing hydrophobicity and volume of the side amino acid residue, locali­zed in the sub-centers thrombin S2 and S3 were carried out. The kinetic parameters of individual stages of the enzymatic reaction process (Ks, k2, k3) were estimated. It is shown that the efficiency of acylation and deacylation stages of the enzymatic reaction decreases with increasing hydrophobicity of the substituent in P2 as well as P3, at the same time the affinity of selected peptides toward enzyme is steadily increasing.
With the aim to evaluate the hydrophobicity of compounds a LogP value was calculated and was made an attempt to compare them with the correspondent Ki values. Comparative kinetic analysis of Z-Arg-OMe and its uncharged analogue Z-Cit-OMe has shown the absence of uncharged analog hydrolysis, however, the mentioned citrulline derivate inhibits the hydrolysis of the charged analogue. These findings confirm the important role of hydrophobic moiety in the structure of thrombin inhibitors in preferential binding mode and inhibition of thrombin active side.

Participation of proteinkinase CK2 in regulation of human erythrocytes plasma membrane redox system activity: relative contribution of Са(2+)-dependent and Са(2+)-independent mechanisms of its activation

I. N. Iakovenko, V. V. Zhirnov, A. P. Kozachenko,
O. V. Shablykin, V. S. Brovarets

Institute of Bioorganic and Petroleum Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: brovarets@bpci.kiev.ua

Involvement of protein kinase CK2 ( in modulation of live cells trans-plasma membrane electron transport was first discovered. Using human erythrocytes a decrease of plasma membrane redox system (PMRS) activity is shown under the action of specific protein kinase CK2 inhibitors.
Using inhibitory analysis the activity regulation of human erythrocytes PMRS by Ca2+-dependent and Ca2+-independent mechanisms were investigated. It was shown that functional Ca2+-antagonists (nitrendipine and calmidazolium) significantly increased, and functional Ca2+-agonists to some extent reduced (calcimycin) or did not affect (Bay K 8644) the trans-plasma membrane electron transport in these cells. In all cases of calcium signaling the system modifications inhibition of kinase CK2 is also accompanied by significant reductions of erythrocytes PMRS activity. Against the background of kinase CK2 inhibition the mentioned influence activity of nitrendipine and Bay K 8644 on PMRS does not change, that of calmidazolium increases and of calcimycin is not revealed. Obtained results demonstrate the inhibitory effect of Ca2+ and calmodulin on human erythrocytes PMRS and the activation of this system by protein kinase CK2 mainly through Ca2+-independent mechanisms. Some participation of calmodulin in PMRS activity regulation by kinase CK2 is discussed.

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

H. V. Danylovych

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+-dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

Inhibition of fibrin polymerization by synthetic peptides corresponding to Аα195-205 and γ69-77 sites of fibrin molecule

T. A. Pozniak, L. P. Urvant, P. G. Gritsenko, V. I. Chernishov,
N. A. Pydiura, E. V. Lugovskoi, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: lougovskoy@yahoo.com

Using the idea of “proline brackets” we have found four sites in fibrin amino acid sequence, and appropriate peptides were synthesized: γ69NPDESSKPN77, Bβ228QPDSSVKPY236, Bβ455RPFFPQ460 and Aα195LPSRDRQHLPL205. Turbidity and electron-microscopy analyses have demonstrated that synthetic peptide Аα195-205 specifically inhibited the stage of fibrin protofibril formation and peptide γ69-77 – the stage of fibrin protofibril lateral association. The data obtained testify that there are the sites involved in these processes in the appropriate amino acid sequences of fibrin molecule.