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.

[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.

[Study on secondary structure and properties of alpha- and gamma-forms of human thrombin].

Secondary structure and enzymatic properties of human a-thrombin and its gamma-form (obtaining during autolysis of the native enzyme) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). According to DSC-data both alpha-thrombin and gamma-thrombin contained only one thermal transition peak at 58.5 and 53.3 degrees C, respectively. A comparison of these values suggested that gamma-form is less stable than initial a-thrombin. In contrast to that the thermogram of DIP-a-thrombin had two peaks (57.5 and 64.5 degrees C). CD spectra showed that conversion a- to gamma-thrombin influenced the secondary structure of the enzyme slightly. The study of the inhibitory effect of such polyanions as ATP and dextran sulfate (DS) upon thrombin-catalyzed cleavages of fibrinogen has shown that the growth of the negative charge of the polyanion molecule resulted in the increase of its inhibitory activity. The catalytically non-active DIP-alpha-thrombin, which retained the native anion-binding exosite 1, was shown to decrease the inhibitory power of the dextran sulfate. It was explained by competition of DS with the exosite 1 of both alpha- and DIP-alpha -thrombin. In contrast to that DIP-gamma-thrombin having exosite 1 destroyed neither competed nor influenced the anticoagulant capacity of dextran sulfate toward the native alpha-thrombin. In accordance with our data thrombin consists of two rather strong interacting domains. It was shown further that its anion-binding exosite 1 may play a significant role in the interaction of the enzyme with dextran sulfate.

[The synthesis of arginine derivatives of chromone and azauracil].

The coupling of N-succinimide esters of 3-[7-hydroxy-3-(4-methyl-1,3-thiazol-2-yl)-6-ethyl-4-oxo-4H-chromen-2-yl]propanoic acid and 5-carboxymethyl-6-azauracil with free arginine yielded the corresponding arginine derivatives, which were purified by crystallization. The structures of the compounds were confirmed by 1H NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

[Synthesis and antithrombin activity of phosphoalanine-containing peptides]. / Sintez i uzuchenie antitrombinovoi aktivnosti pepidov, soderzhashchikh fosfoalanin.

Boc/Tos-L-Phe-L-Arg-Xaa tripeptides (where Xaa = L-Ala-OBut, L-Ala, or DL-AlaP (OC2H5)2) were synthesized by conventional methods of peptide synthesis in solution. Special features of their interaction with thrombin and trypsin were studied. Unlike trypsin, thrombin did not catalyze the hydrolysis of the L-Arg-L-AlaP-(OC2H5)2 bond. The Tos-L-Phe-L-Arg-DL-AlaP(OC2H5)2 peptide was the most active inhibitor of thrombin among all the compounds studied. The relationship between the structure and inhibitory action of the synthesized peptides is discussed. A part of this study was reported in the Augustusburg Conference of Advanced Science: Nucleic Acids--Targets and Tools, September 17-19, 2000, Germany.

[Kinetics of trypsin-catalyzed hydrolysis of some arginine-containing peptide methyl esters]. / Kinetika gidroliza tripsinom metilovykh éfirov nekotorykh argininsoderzhashchikh peptidov.

The kinetics of trypsin-catalyzed hydrolysis (at pH 8.5) of methyl esters of some synthetic dipeptides containing the residues of both arginine and L(D)-p-fluorophenylalanine or L(D)-tyrosine has been studied. The digestion of Tos-(pF)Phe-Arg-OMe was shown as unfollowing the Michaelis-Menten kinetic since in the reaction course the substrate activation is observed and while the reaction product is the enzymatic process inhibitor. In contrast to this, the hydrolysis of other substrates studied, follows the normal Michaelis-Menten kinetic.

Solid-state conjugation of proteins with hydrophobic compounds in non-denaturing conditions. I. Acylation of proteins by dansyl proline using a polymeric N-hydroxysuccinimide ester.

A method of conjugating protein molecules under native conditions with water-insoluble hydrophobic compounds is developed. It permits very water-insoluble acids to be gently coupled to the primary amines on proteins or other biopolymers. For this purpose we synthesized a polymer (co-polymer of N-hydroxymaleimide and N-vinylpyrrolidone cross-linked by benzidine) which swells equally well in water and in organic solvents. Hydrophobic substances are first activated by esterification to this polymer in organic solvent and then conjugated to protein by acylation in aqueous medium at pH 8.0-8.5. Thus, the contact of native protein with organic co-solvent may be completely avoided. The application of this approach is demonstrated by labeling trypsinogen and plasminogen with dansyl proline.

[Synthesis and study of biological activity of stereoisomeric dipeptides containing tyrosine and arginine residues]. / Sintez i issledovanie biologicheskoi aktivnosti stereoizomernykh dipeptidov, soderzhashchikh ostatki tirozina i arginina.

Series of methyl esters of stereoisomeric dipeptides of the sequences Tyr-Arg and Arg-Tyr has been synthesized by classic methods of the peptide chemistry. The study of their reactivity towards thrombin and trypsin has shown that the kinetic parameters of enzyme-catalyzed hydrolyses of stereoisomeric compounds differ in values essentially. Testing the synthetic peptides on analgic effect, on inhibition of the reaction fibrinogen with thrombin or on influence upon the process of fibrin-monomer polymerization has shown that these biological effects depend on peptide structure and on configuration of amino acid residues forming the peptides.

[Natural and synthetic inhibitors of thrombin. I. Natural inhibitors]. / Prirodnye i sinteticheskie ingibitory trombina. I. Prirodnye ingibitory.

Structures and properties of main physiological (heparin, antithrombin III, heparin cofactor II) and nonphysiological (hirudin, thrombin-binding aptamers, cyclotheonamides) natural thrombin inhibitors and its fragments and synthetic analogs (hirugen, hirulogs, hirunorms, pentasaccharides, macrocyclic alpha-keto amides) were reviewed. The molecular mechanisms of interaction of these compounds with thrombin and their anticoagulant activity at preclinical and clinical trials are discussed. The examined of natural thrombin inhibitors and their synthetic fragments and analogs are perspective for prophylaxis and treatment of different thrombo-embolic diseases.

[Natural and synthetic inhibitors of thrombin. II. Synthetic low-molecular-weight inhibitors]. / Prirodnye i sinteticheskie ingibitory trombina. II. Sinteticheskie nizkovmolekuliarnye ingibitory.

The up-to-date problems, concerning the structure and properties of two types of inhibitors are reviewed. It is particularly considered properties of low-molecular weight thrombin inhibitors that have electrophilic groups capable to react with Ser-195 of thrombin (peptidyl-chloromethyl ketones, aldehydes, ketomethylene derivatives and derivatives of boric and phosphoric acids) and the competitive reversible thrombin inhibitors. The review focuses on methods of modification of the structure in the natural inhibitors and design of new peptidomimetics. The prospects for prophylaxis and treatment of diverse thromboembolic diseases are discussed.

[New donor-acceptor pairs for fluorogenic substrates with intramolecular fluorescence energy transfer for thrombin and trypsin]. / Novye donorno-aktseptornye pary dlia fluorogennykh substratov s vnutrimolekuliarnym perenosom énergii fluorestsetsii dlia trombina i tripsina.

New substrates for thrombin and trypsin are described: a fluorogenic substrate Abz-Pro-Arg-Gly-Nph (I), whose action is based on intramolecular fluorescence energy transfer, and H-D-Trp-Pro-Arg-pNA (II), which can be used both as a chromogenic substrate and as a substrate with the intramolecular fluorescence energy transfer. In substrate (I), a 4-nitrophenylhydrazide group was first used as an acceptor of excitation energy of the 2-aminobenzoyl group. The substrate is poorly hydrolyzed by thrombin (kcat/K(m) = 1.4 x 10(3) M-1 s-1) and is efficiently cleaved by trypsin (kcat/K(m) = 3.15 x 10(6) M-1 s-1). The hydrolysis of (II) can be monitored both spectrophotometrically, by absorbance at 405 nm, and from the increase in fluorescence at 340 nm. In the efficiency of hydrolysis with thrombin (kcat/K(m) = 3.0 x 10(6) M-1 s-1), compound (II) is comparable with the known chromogenic substrates for this enzyme. The proposed donor-acceptor pairs are promising in designing substrates with the intramolecular fluorescence energy transfer for a variety of proteolytic enzymes.

Synthesis and study on a novel inhibitor of thrombin containing no side chain at the P1 position of the polypeptide chain.

The presented work allows one to speculate that the hydrophobic contacts of the residues located at the P2 and P3 positions with the corresponding subsites of thrombin (S2 and S3) allow the synthesis of compounds that can react with thrombin specifically and probably may not interact with other trypsin-like serine proteases. Substitution of proline by m-Abz-residue may result in the development of novel substrates and inhibitors for thrombin.

[Thrombin substrates containing aminobenzoic acids]. / Sybstraty trombina, soderzhashchie aminobenzoinye kisloty.

In order to study the interactions of synthetic substrates with the active site of thrombin the compounds of R-CO-Abz-Arg-OMe were obtained which contained, o-, m- and p-amino benzoic acids (Abz) at P2-subsite. R-CO-moiety was butyric, perfluorobutyric, benzoyl, 2-fluorobenzoyl and perfluorobenzoyl groups. Kinetic parameters of their hydrolysis by thrombin were measured and antithrombin activity was studied. Introduction of fluorine atoms into N-acyl groups caused insignificant alterations of the substrate or inhibitor properties of the compounds containing N-butyric group, but significantly influenced the substrate properties, when peptides contained N-benzoyl group. The interaction mechanism of the obtained substrates with thrombin was proposed.

Similarity of the effects of tosyl-L-arginine methyl ester, atropine, caffeine and antitumour alkylating agent on some biological functions of thrombin and platelet 12-lipoxygenase.

The effect of the synthetic thrombin substrate (TAME) and three compounds exerting an opposite effect on Ca-PPI and AdC (caffeine, atropine and meta-tolyl derivative of mechlorethamine (TDM)) on hormone-like and catalytic functions of thrombin was studied. It is shown that both TAME and other drugs under test block effectively the thrombin-induced platelet aggregation, as well as protect the active site of the enzyme from denaturation by dithiothreitol. The same compounds inhibit thrombin in thrombin-fibrinogen reaction and platelet 12-lipoxygenase. These data suggest identity of thrombin moieties which determine its enzymatic and hormone-like activities.