Chemical structure and properties of low-molecular furin inhibitors.

The review is devoted to the analysis of the relationship between a chemical structure and properties of low-molecular weight inhibitors of furin, the most studied proprotein convertase, which is involved in the development of some pathologies, such as oncologic diseases, viral and bacterial infections, etc. The latest data concerning the influence of peptides, pseudo-peptides, aromatic and heterocyclic compounds, some natural ones such as flavonoids, coumarins, and others on enzyme inactivation are considered. The power of furin inhibition is shown to rise with the increasing number of positively charged groups in the structure of these compounds. Peptidomimetics (Ki = 5-8 pM) are shown to be the most effective furin inhibitors. The synthesized substances, however, have not been used in practical application yet. Nowadays it is very important to find more selective inhibitors, improve their stability, bioavailability and safety for the human organism.

FURIN INHIBITORS BASED ON THE DERIVATIVES OF CALIX[4]ARENE CX3im.

The aim of this work was to study antifurin activity of some derivatives of calix[4]arenes modified on the upper rim of the macrocycle by positively charged or uncharged groups. It was found that calixarene CX3im derivatives containing positively charged N-methylimidazolium cycles were indeed able to inhibit furin (K(i) = 58.2 µM). The magnitude of the effects depended also on the hydrophobicity of the substituents located on the lower rim of the macrocycle. The findings indicated the possibility of creating furin inhibitors of new generation based on the calix[4]arene platform.

Synthesis, biological evaluation and docking of novel bisamidinohydrazones as non-peptide inhibitors of furin.

A series of novel non-peptidicfurin inhibitors with values of inhibitory constants (Ki) in the range of 0.74-1.54 µM was obtained by interactions of aminoguanidine hydrocarbonate with three diaryldicarbalde- hydes. Correspondingly p-hydroquinone, piperazine and adipic acid were used as linkers between their ben- zene moieties. Docking studies of these new inhibitors into recently published 3D-structure of human furin (PDB code 4OMC) showed that they were able to interact with subsites S1 and S4 of the enzyme. The overall arrangement of bisamidinohydrazones into furin active site was similar to the position of the ligand co- crystallized with a protease. Observations obtained with molecular modeling allowed further guidance into chemical modifications of the synthesized inhibitors which improve their inhibitory activity.

[Non-peptide furin inhibitors based on amidinohydrazones of diarylaldehydes].

A series of novel non-peptidic furin inhibitors containing amidinohydrazone moieties has been synthesized under interaction of dialdehydes, the derivatives of ethylene diethylvanillin ethers, with aminoguanidine bicarbonate. Two aryl cycles were bridged by 1,2-ethylene-, 1,4-buthylene- or 1,4-dimethylenebenzene-group. The compounds have been found to inhibit furin. The antifurin activity was shown to grow with the increase of the length and/or hydrophobicity of the bridge. The most potent compound, containing in the bridge the lypophylic benzene cycle was found to inhibit the activity of furin with Ki = 0.51 microM.

[Estimation of ROS in the presence of biologically active substances by porous silicon fluorescence].

The fluorescence spectra of the porous silicon modified by water solutions of biologically active materials and materials of biological origin are recorded as well as the fluorescence spectra of the porous silicon modified by lecithin monolayers grown on the surface of water solutions of the biologically active materials. The analysis of the obtained spectra made it possible to conclude on the effect of the studied materials on the content of ROS.

[Structure and properties of proprotein convertase inhibitors].

This review is devoted to structure and properties of proprotein convertases (PCs), the intracellular Ca(2+)-dependent serine endoproteases of mammalia, that play the essential role in the processing of inactive protein precursors and their transforming into bioactive mature products. PCs are also implicated in development of a great variety of diseases including bacterial or viral infections and such pathologies as cancer, Alzheimer's disease, obesity and so on. Owing to these findings, PCs are considered as promising targets for design of their inhibitors and development of new potential therapeutic agents. Only several endogenous protein inhibitors are identified now for PCs: pro7B2 (Proprotein 7B2), the specific chaperon of PC2, granine-like precursor of neuroendocrine protein proSAAS, the selective ligand of PC1, and serpin Spn4A (Serine Proteinase Inhibitor) of Drosophila melanogaster that inhibits PC2 and furin. By the methods of site-directed mutagenesis, the bioengineered inhibitors of PCs were also designed. Structures and properties of protein or peptide fragments as inhibitors of PCs were also discussed. Particularly, the properties of polyarginines and small peptides containing pseudopeptide bond at the scissile site a suitable peptide substrate were described. The inhibitory activity of non-peptide compounds such as derivatives of andrographolid from Andrographis paniculata (K(i) = 2.6-200 microM against furin), certain complexes of pyridine analogs with ions of Cu2+ or Zn2+ inhibiting furin with IC50 = 5-10 microM, derivatives of 2,5-dideoxy-streptamine containing several guanidine groups (K(i) = 6-812 nM for furin) and also a number of dicoumarols (K(i) = 1-185 microM against furin) and some flavonoids (with K(i) = 5-230 microM for furin) were reflected in the article. The effects of enediynyl-amino acids derivatives or their peptides (K(i) = 40 nM against furin) were considered. Inhibition of PC2 by N-acylated bicyclic guanidines (K(i) = 3.3-10 microM) or derivatives of pyrrolidin bispyperazines (K(i) = 0.54-10 microM) are considered too. Some of synthesized derivatives may serve as lead compounds for design of the specific inhibitors for individual PCs.

[Study on derivatives of 5-amino-4-acylamino-1H-pyrazole as inhibitors of furin].

A series of 5-amino-1H-pyrazoles was synthesized and studied as inhibitors of furin. The most potent compound, 5-amino-4-acetylamino-3-(4-methylphenylamino)1H-pyrazole, was found to retard the activity of furin by mixed-type inhibition with K = 288 microM. These findings permit to plan new ways for chemical modifications of the 5-amino-1H-pyrazole structure and design more potent furin inhibitors of non-peptide nature.

[New non-peptide inhibitors of furin].

Furin, a human subtilisin-related proprotein convertase, is the most important pharmaceutical target because it plays a vital role in development of numerous disease processes. To identify a new class of small non-peptide inhibitors of furin we performed a study of several flavonoids and some natural products. Glycosylated flavonoids: rutin, naringin, baikalin and methylhesperidin were shown to inhibit furin at pH 7.2 reversibly and competitively with Ki- 80-200 microM. The Ki values were derived from Dixon and/or Eadie-Hofstee plots using fluorogenic substrate Boc-Arg-Val-Arg-Arg-AMC. Although studied flavonoids display only a temperate furin inhibition, they may serve as a great potential for the future development of more potent non-peptide inhibitors against furin.

[Furin and its biological role].

The survey deals with structure, properties and biological role of furin, the calcium-dependent serine endoprotease, which is expressed in all tissues and cell lines examined. It is the best-characterized representative of the mammalian subtilisin-like family of proprotein convertases, which is capable to cleave precursors of a wide variety of proteins, including hormones, growth factors, serum proteins, proteases of the blood-clotting system, matrix metalloproteinases, receptors, viral envelope glycoproteins, and bacterial exotoxins, and so on. The enzyme plays the essential role in embryogenesis, homeostasis; it is also involved in tumor metastasis, activation and virulence of many bacterial and viral pathogens and in neurodegenerative processes associated with Alzheimer's disease. Furin is a very specific enzyme: it recognizes the cleavage-site sequence Arg-Xaa-Lys/Arg-Arg and catalyzes the hydrolysis of the precursors, containing a pair of basic amino acids Arg-Arg or Lys-Arg. The enzyme is a multidomain protein which is initially synthesized as 100 kDa glycosylated profurin consisting of 794 amino acid residues (for human furin) and including a N-terminal signal peptide, propeptide, catalytic domain, possessing approximately 30% homology to subtilisin, a well-conserved P-domain, Cys-rich domain, transmembrane domain and cytoplasmic domain. The active site cleft of furin differs considerably from that of subtilisin with respect to the depth, shape and molecule charge. In furin it is a canyon-like groove with clusters of negatively charged residues along it. Furin inhibitors are rather promising therapeutic agents for treating furin-mediated diseases and bacterial infections. Most furin inhibitors belong to proteins or peptides. The protein-based inhibitors include both naturally occurring and bioengineered variants of protease inhibitors. The peptide-based inhibitors are represented by polyarginines, peptidyl chloromethyl ketones, aminomethyl ketones or ketomethylene pseudopeptides, isostere-containing cyclic peptides, short peptides derived from the prosegments of furin or al-PDX-related peptides. The nonpeptide inhibitors are natural products of the andrographolide family, certain metal complexes of pyridine derivatives and small molecules derived from 2.5-dideoxystreptamine. The furin inhibitors may be used not only as valuable tools for studying furin action, but also as therapeutic agents for furin-dependent diseases.

[The effect of various forms of thrombin on nonspecific high molecular weight substrates]. / Deistvie razlichnykh form trombina na nespetsificheskie vysokomolekuliarnye substraty.

The ability of native alpha- and non-coagulating gamma-thrombin to catalyze the hydrolysis of nonspecific high molecular weight substrates was studied using chymotrypsinogen and the oxidized insulin B-chain as substrates. The effect of thrombin on chymotrypsinogen was estimated by the appearance of caseinolytic activity measured by the increase in the number of terminal NH2-groups in the 2,4,6-trinitrobenzol sulfonic acid reaction. The same reaction was used to study the hydrolysis of insulin by thrombin. It was found that the destruction of the additional center necessary for fibrinogen proteolysis during the alpha-thrombin conversion to the gamma-form did not affect the enzyme ability to hydrolyze nonspecific protein substrates. It was assumed that the low efficiency of non-physiological high molecular weight substrate hydrolysis by thrombin is due to the lack of specific remote interactions in the regulatory site outside the enzyme active center.

[Enzymatic properties of the isolated B-chain of human thrombin]. / Fermentativnye svoistva izolirovannoi B-tsepi trombina cheloveka.

The A- and B-chains have been isolated from the non-covalent complex of human thrombin A- and B-chains, using selective reduction of the interchain disulfide bridge. The B-chain thus isolated (de-A-thrombin) retains its conformation, which is close to the native one and thus differs considerably from the B-chain isolated from the fully reduced enzyme. Nevertheless, the proteolytic (in terms of fibrinogen clotting) and amidase activities of de-A-thrombin are markedly reduced as compared to the native enzyme and the non-covalent complex of A- and B-chains. It is assumed that the A-chain of thrombin is necessary for normal functioning of the active site of thrombin localized in the B-chain.

[Effect of reduction of the interchain disulfide bridge of human thrombin on its enzymatic activity and structure]. / Vliianie vosstanovleniia mezhtsepochechnogo disul'fidnogo mostika trombina cheloveka na ego fermentativnuiu aktivnost' i strukturu.

It was shown that selective hydrolysis of the disulfide bridge between the A- and B-chains of human thrombin in the absence of denaturating agents decrease its proteolytic (e.g., fibrinogen-binding), esterase and amidase activities. Both chains remain bound by non-covalent interactions. A preparation of partially reduced thrombin was obtained and its kinetic parameters were determined. The experimental results suggest that the S-S bond connecting the A- and B-chains of thrombin is involved in the stabilization of the enzyme active center.

[The use of fluorescence spectroscopy for identification of A- and B-thrombin chains during their chromatographic separation]. / Primenenie fluorestsentnoi spektroskopii dlia identifikatsii A- i B-tsepei trombina pri ikh khromatograficheskom razdelenii.

The separation of A- and B-chains of human thrombin has been performed by gel filtration on Sephadex G-100 under the reduction of disulphide bonds with dithiothreitol. Identification of A- and B-chains has been provided by measurements of the fluorescence intensity of fractions at 310 nm and 350 nm which are near the maximum positions of tyrosine and tryptophan fluorescence, respectively. The appearance of A-chain was monitored by an increase of the ratio of Ifl310/Ifl350 greater than 2. The fluorescence spectrum of A-chain has maximum position at 304 nm, which is characteristic of tyrosine fluorescence. The fluorescence spectrum of B-chain has maximum position at 347.5 nm which corresponds to fluorescence of tryptophan residues. The identification of A- and B-chains has been confirmed by the gel electrophoresis data.

[Effect of partial reduction of disulfide bonds on the enzymatic activity of thrombin]. / Vliianie chastichnogo vosstanovleniia disul'fidnykh sviazei na fermentativnuiu aktivnost' trombina.

It was demonstrated that partial reduction of disulfide bonds in thrombin by dithiothreitol in the absence of denaturating agents leads to a decrease of enzymatic activity with respect to fibrinogen coagulation and tosylarginine methyl ester hydrolysis. Polyacrylamide gel electrophoresis and determination of the number of SH-groups liberated in the course of reduction suggest that the observed inactivation is primarily due to the disruption of the S-S-bridge between the A- and B-chains of thrombin.

[The effect of dithiothreitol on the thrombin coagulating activity]. / Vliianie ditiotreitola na svertyvaiushchuiu aktivnost' trombina.

Partial dithiothreitol-reduction of the disulphide thrombin bonds when denaturating agents are absent lowers significantly enzymic activity of thrombin relative to fibrinogen coagulation. This permits supposing that at least one of the disulphide bridges in a thrombin molecule is necessary for stabilization of the space structure important for a specific hydrolysis of fibrinogen.