Green tea polyphenols as inhibitors of ribonuclease A

Ribonucleases (RNases), which are essential for cleavage of RNA, may be cytotoxic due to undesired cleavage of RNA in the cell. The quest for small molecule inhibitors of members of the ribonuclease superfamily has become indispensable with a growing number exhibiting unusual biological properties. Thus, inhibitors of RNases may serve as potential drug candidates. Green tea catechins (GTC), particularly its major constituent (-)-epigallocatechin-3-gallate (EGCG), have reported potential against cell proliferation and angiogenesis induced by several growth factors including angiogenin, a member of the RNase superfamily. This study reports the inhibition of bovine pancreatic ribonuclease A (RNase A) by EGCG and GTC. This has been checked qualitatively by an agarose gel based assay. Enzyme kinetic studies with cytidine 2',3' cyclic monophosphate as the substrate have also been conducted. Results indicate substantial inhibitory activity of a noncompetitive nature with an inhibition constant of approximately 80 microM for EGCG and approximately 100 microM for GTC measured in gallic acid equivalents.     

Inhibition of ribonuclease A by nucleoside–dibasic acid conjugates

We report the inhibition of the ribonucleolytic activity of ribonuclease A (RNase A) by nucleoside–dibasic acid conjugates for the first time. Agarose gel and precipitation assays show that the spacer length and the pK_a of the carboxylic group have an important role in the inhibitory capacity. Kinetic experiments indicate a competitive mode of inhibition with inhibition constant (K_i) value of 132 ± 3 μM for Oxa-aT. Docking studies revealed that the carboxylic group of the most active compounds is within hydrogen bonding distance of His-12, Lys-41 and His-119.     

Carbocyclic pyrimidine nucleosides as inhibitors of S-adenosylhomocysteine hydrolase

The design, synthesis, and unexpected inhibitory activity against S-adenosyl-homocysteine (SAH) hydrolase (SAHase, EC 3.3.1.1) for a series of truncated carbocyclic pyrimidine nucleoside analogues is presented. Of the four nucleosides obtained, 10 was found to be active with a Ki value of 5.0 microM against SAHase.     

A study on the positive ellipticity in the circular dichroism of ribonuclease A

The small positive elliplicity near 239 nm in the CD spectrum of RNase has been investigated as a function of pH. Theoretical calculations using CD parameters representing buried or exposed tyrosine residues have been carried out. A comparison of the theoretical calculations with experimental data suggests that the changes in the band's intensity, as a function of pH, arise mainly from electronic transitions associated with the tyrosine residues. The buried tyrosine residues are the major contributors to the ellipticity in this region at neutral pH. At higher pH contributions from exposed residurs are also observed.     

Effects of neutral salts on the circular dichroism spectra of ribonuclease a and ribonuclease s

The circular dichroism (CD) spectra of ribonuclease A, ribonuclease S, and N-acetyltyrosineamide were recorded as a function of pH in the presence of various concentrations of inorganic salts. Above pH 9.0 salting-in of tyrosine residues increases their intramolecular associations. This association enhances the contribution from these residues to the CD spectrum leading to an apparent titration curve that is shifted toward lower pH. The data indicate that unfolding of ribonuclease A and S by inorganic salts does not begin with disrupting existing electrostatic interactions. But, as the unfolding process progresses, disruption of electrostatic interactions may take place. This is consistent with our previous calorimetric studies which suggest that unfolding of ribonuclease A by salts proceeds initially by energetically favorable solvation of the folded protein. An increase in ellipiticity at 275 nm of partially unfolded protein in salt was observed as the pH was changed from 7.0 to 4.0. This observation may suggest that the isothermal unfolding of the protein by salts at low pH proceeds through an intermediate step which involves histidine residues and causes a conformational change in the tyrosine's asymmetric environment.     

The compactness of ribonuclease A and reduced ribonuclease A

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small-angle X-ray scattering. The R_g values and the Kratky plots showed that non-reduced ribonuclease A maintain a compact shape with a R_g value of about 17.3 Å in 8 M urea. The reduced ribonuclease A is more expanded, its R_g value is about 20 Å in 50 mM Tris-HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.     

Structurally diverse 5-substituted pyrimidine nucleosides as inhibitors of Leishmania donovani promastigotes in vitro

The following structurally diverse 5-substituted-2'-deoxyuridine nucleosides displayed potent in vitro antileishmanial activity: 5-formyl, 5-(2,2,-dicyanovinyl)-, 5-(2-cyano-2-ethoxycarbonylvinyl), 5-(2-cyano-2-methoxycarbonylvinyl)-, 5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)- and related congeners, and the 5-(3-methyl-5-oxo-1-phenyl-4,5-dihydro-4H-pyrazol-4-ylidene) group.     

1,5-Disubstituted 1,2,3-triazole linked disaccharides: Metal-free syntheses and screening of a new class of ribonuclease A inhibitors

1,5-Regioisomeric triazole linked disaccharides have been synthesized and screened for their inhibitory properties against ribonuclease A (RNase A). The angular constraint-driven ‘crescent shaped’ inhibitors accommodated themselves into the enzyme active site. An improved enzyme inhibition was observed with increased H-bonding ability of polar functional groups in the modified disaccharides. In this series, introduction of two carboxyl groups in the furanose rings elicited the best result with an inhibition constant of 50?±?3?µM. This is the first ever report on the use of disaccharides as RNase A inhibitors.     

Triazole double-headed ribonucleosides as inhibitors of eosinophil derived neurotoxin

Eosinophil derived neurotoxin (EDN) is an eosinophil secretion protein and a member of the Ribonuclease A (RNase A) superfamily involved in the immune response system and inflammatory disorders. The pathological actions of EDN are strongly dependent on the enzymatic activity and therefore, it is of significant interest to discover potent and specific inhibitors of EDN. In this framework we have assessed the inhibitory potency of triazole double-headed ribonucleosides. We present here an efficient method for the heterologous production and purification of EDN together with the synthesis of nucleosides and their biochemical evaluation in RNase A and EDN. Two groups of double-headed nucleosides were synthesized by the attachment of a purine or a pyrimidine base, through a triazole group at the 3′-C position of a pyrimidine or a purine ribonucleoside, respectively. Based on previous data with mononucleosides these compounds were expected to improve the inhibitory potency for RNase A and specificity for EDN. Kinetics data revealed that despite the rational, all but one, double-headed ribonucleosides were less potent than the respective mononucleosides while they were also more specific for ribonuclease A than for EDN. Compound 11c (9-[3′-[4-[(cytosine-1-yl)methyl]-1,2,3-triazol-1-yl]-β-d-ribofuranosyl]adenine) displayed a stronger preference for EDN than for ribonuclease A and a K_i value of 58 μM. This is the first time that an inhibitor is reported to have a better potency for EDN than for RNase A. The crystal structure of EDN–11c complex reveals the structural basis of its potency and selectivity providing important guidelines for future structure-based inhibitor design efforts.     

Compensating effects on the cytotoxicity of ribonuclease A variants

Ribonuclease (RNase) A can be endowed with cytotoxic activity by enabling it to evade the cytosolic ribonuclease inhibitor protein (RI). Enhancing its conformational stability can increase further its cytotoxicity. Herein, the A4C/K41R/G88R/V118C variant of RNase A was created to integrate four individual changes that greatly decrease RI affinity (K41R/G88R) and increase conformational stability (A4C/V118C). Yet, the variant suffers a decrease in ribonucleolytic activity and is only as potent a cytotoxin as its precursors. Thus, individual changes that increase cytotoxicity can have offsetting consequences. Overall, cytotoxicity correlates well with the maintenance of ribonucleolytic activity in the presence of RI. The parameter (k(cat)/K(m))(cyto), which reports on the ability of a ribonuclease to manifest its ribonucleolytic activity in the cytosol, is especially useful in predicting the cytotoxicity of an RNase A variant.     

Refolding of ribonuclease A monitored by real-time photo-CIDNP NMR spectroscopy

Photo-CIDNP NMR spectroscopy is a powerful method for investigating the solvent accessibility of histidine, tyrosine and tryptophan residues in a protein. When coupled to real-time NMR, this technique allows changes in the environments of these residues to be used as a probe of protein folding. In this paper we describe experiments performed to monitor the refolding of ribonuclease A following dilution from a high concentration of chemical denaturant. These experiments provide a good example of the utility of this technique which provides information that is difficult to obtain by other biophysical methods. Real-time photo-CIDNP measurements yield residue-specific kinetic data pertaining to the folding reaction, interpreted in terms of current knowledge of the folding of bovine pancreatic ribonuclease A.     

An evidence for the equilibrium unfolding intermediates of ribonuclease A by tritium labeling method

A formation of a molten globule in the unfolding of ribonuclease A could be considered as an evidence supporting a hypothesis on the existence of such intermediates on the pathway of a protein folding. Using a novel technique (tritium labeling method) we have showed that the ribonuclease A equilibrium unfolding in urea and guanidinium chloride (GuCl) solutions proceeds through a formation of intermediates whose properties (compactness, retention of the larger part hydrophobic core, secondary structure, and native-like folding pattern) correspond to the fundamental characteristics of the molten globule state. The both intermediates are the “wet” molten globules (the globule interior contains the water molecules). The results reveal the noticeable distinctions in intermediates structure, first of all, in the extent of their compactness. The urea intermediate is less compact than that in GuCl. It is shown that the refolding of the protein denatured by GuCl results in the formation of the intermediate which enzyme activity is virtually the same as the activity of the native protein.     

Triazole pyrimidine nucleosides as inhibitors of Ribonuclease A. Synthesis,biochemical, and structural evaluation

Five ribofuranosyl pyrimidine nucleosides and their corresponding 1,2,3-triazole derivatives have been synthesized and characterized. Their inhibitory action to Ribonuclease A has been studied by biochemical analysis and X-ray crystallography. These compounds are potent competitive inhibitors of RNase A with low μM inhibition constant (K_i) values with the ones having a triazolo linker being more potent than the ones without. The most potent of these is 1-[(β-d-ribofuranosyl)-1,2,3-triazol-4-yl]uracil being with K_i = 1.6 μM. The high resolution X-ray crystal structures of the RNase A in complex with three most potent inhibitors of these inhibitors have shown that they bind at the enzyme catalytic cleft with the pyrimidine nucleobase at the B₁ subsite while the triazole moiety binds at the main subsite P₁, where P-O5′ bond cleavage occurs, and the ribose at the interface between subsites P₁ and P₀ exploiting interactions with residues from both subsites. The effect of a susbsituent group at the 5-pyrimidine position at the inhibitory potency has been also examined and results show that any addition at this position leads to a less efficient inhibitor. Comparative structural analysis of these RNase A complexes with other similar RNase A—ligand complexes reveals that the triazole moiety interactions with the protein form the structural basis of their increased potency. The insertion of a triazole linker between the pyrimidine base and the ribose forms the starting point for further improvement of these inhibitors in the quest for potent ribonucleolytic inhibitors with pharmaceutical potential.     

Differences in the denaturation behavior of ribonuclease A induced by temperature and guanidine hydrochloride

Moderate temperatures or low concentrations of denaturants diminish the catalytic activity of some enzymes before spectroscopic methods indicate protein unfolding. To discriminate between possible reasons for the inactivation of ribonuclease A, we investigated the influence of temperature and guanidine hydrochloride on its proteolytic susceptibility to proteinase K by determining the proteolytic rate constants and fragment patterns. The results were related to changes of activity and spectroscopic properties of ribonuclease A. With thermal denaturation, the changes in activity and in the rate constants of proteolytic degradation coincide and occur slightly before the spectroscopically observable transition. In the case of guanidine hydrochloride-induced denaturation, however, proteolytic resistance of ribonuclease A initially increases accompanied by a drastic activity decrease far before unfolding of the protein is detected by spectroscopy or proteolysis. In addition to ionic effects, a tightening of the protein structure at low guanidine hydrochloride concentrations is suggested to be responsible for ribonuclease A inactivation.     

The exo- or endonucleolytic preference of bovine pancreatic ribonuclease A depends on its subsites structure and on the substrate size

The cleavage pattern of oligocytidylic acid substrates by bovine pancreatic ribonuclease A (RNase A) was studied by means of reversed-phase HPLC. Oligocytidylic acids, ranging from dinucleotides to heptanucleotides, were obtained by RNase A digestion of poly(C). They were identified by MALDI-TOF mass spectrometry; it was confirmed that all of them corresponded to the general structure (Cp)(n)C>p, in which C>p indicates a 2',3'-cyclic phosphate. This is a confirmation of the proposed mechanism for RNase A, wherein the so-called hydrolytic (or second) step is in fact a special case of the reverse of transphosphorylation (first step). The patterns of cleavage for the oligonucleotide substrates show that the native enzyme has no special preference for endonucleolytic or exonucleolytic cleavage, whereas a mutant of the enzyme (K7Q/R10Q-RNase A) lacking p(2) (a phosphate binding subsite adjacent, on the 3' side, to the main phosphate binding site p(1)) shows a clear exonucleolytic pattern; a mutant (K66Q-RNase A) lacking p(0) (a phosphate binding subsite adjacent, on the 5' side, to the main phosphate binding site p(1)) shows a more endonucleolytic pattern. This indicates the important role played by the subsites on the preference for the bond cleaved. Molecular modeling shows that, in the case of the p(2) mutant, the amide group of glutamine can form a hydrogen bond with the 2',3'-cyclic terminal phosphate, whereas the distance to a 3',5'-phosphodiester bond is too long to form such a hydrogen bond. This could explain the preference for exonucleolytic cleavage shown by the p(2) mutant.     

Preparation and biological evaluation of 1′-cyano-2′-C-methyl pyrimidine nucleosides as HCV NS5B polymerase inhibitors

The first synthesis of 1′-cyano-2′-C-methyl pyrimidine nucleosides is described. Anti-HCV activity of these nucleosides and their nucleotide phosphoramidate prodrugs was assessed and compared to the 1′-unsubstituted counterparts and to the related 1′-cyano-2′-C-methyl C-nucleoside parent of GS-6620.     

Purification effect of artificial chaperone in the refolding of recombinant ribonuclease A from inclusion bodies

Artificial chaperone (AC) containing cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD) has been used to refold recombinant ribonuclease A (RNase A) from inclusion bodies (IBs). At low urea concentration (0.8 M), the AC could enhance the refolding yield of RNase A by effectively suppressing its intermolecular interaction-induced aggregation. As a result, 0.9 mg/mL RNase A could be 77% refolded, which was a 57% increase as compared to that without the AC. At high protein concentration range (0.9–2.3 mg/mL in total protein concentrations) and 1.6 M urea, CTAB selectively precipitated contaminant proteins distinctly, so a purification effect was achieved. For example, 1.5 mg/mL RNase A could be 62% refolded and recovered at a purity of 87%, which was a 34% increase in purity as compared to that in IBs (65%). The precipitation selectivity was considered due to the differences in the hydrophobicity of the proteins. The work indicates that by using the AC, RNase A could be efficiently refolded at low urea concentration and purified at high urea concentration from IBs at high protein concentrations.     

A ribonuclease with distinctive features from the wild green-headed mushroom Russulus virescens

A ribonuclease with an N-terminal sequence different from those of other ribonucleases has been purified from fruiting bodies of the mushroom Russula virescens. The RNase was adsorbed on DEAE-cellulose and Q-Sepharose in 10mM Tris-HCl buffer (pH 7.1-7.3) and on CM-Sepharose in 10mM NH(4)OAc buffer (pH 4.6), unlike other mushroom ribonucleases which are unadsorbed on DEAE-cellulose. The RNase demonstrated a molecular mass of 28kDa in both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to other mushroom ribonucleases which are monospecific, it exhibited co-specificity towards poly A and poly C. It demonstrated a pH optimum of 4.5, which is lower than values reported for other mushroom ribonucleases, and a temperature optimum of 60 degrees C.     

A ribonuclease from Chinese ginseng (Panax ginseng) flowers

A ribonuclease, with a molecular mass of 23kDa, and much higher activity toward poly(U) than poly(C) and only negligible activity toward poly(A) and poly(G), was isolated from the aqueous extract of Chinese ginseng (Panax ginseng) flowers. The ribonuclease was unadsorbed on diethylaminoethyl-cellulose and adsorbed on Affi-gel blue gel and carboxymethyl-cellulose. High activity of the ribonuclease was maintained at pH 6-7. On either side of this pH range, there was a precipitous drop in enzyme activity. The activity of the enzyme peaked at 50 degrees C and fell to about 20% of the maximal activity when the temperature was lowered to 20 degrees C or raised to 80 degrees C. The characteristics of this ribonuclease were different from those of ribonuclease previously purified from ginseng roots.