Structure-activity relationship study of steroidal and nonsteroidal inhibitors of the enzyme estrone sulfatase

We report the initial results of a series of molecular modelling studies to investigate the structural properties of non-steroidal inhibitors required for inhibitory activity against the enzyme estrone sulfatase (ES) [the enzyme responsible for the conversion of nonactive (sulfated) estrone to the active (nonsulfated) estrone]. From the results of the present study, we conclude that the C(17) polar group may not be necessary for inhibitory activity and that the only requirement appears to be the mimicking of the steroid C(3) sulfonate group. To test our hypotheses, we have designed novel straight chain inhibitors based upon alkyl alcohols, which upon evaluation, have been shown to possess inhibitory activity (e.g., an inhibitor based upon trichloroethanol has been shown to possess 46% inhibition at 0.76mM).     

A molecular model for the interaction between vorozole and other non-steroidal inhibitors and human cytochrome P450 19 (P450 aromatase)

In a previous study (Vanden Bossche et al., Breast Cancer Res. Treat. 30 (1994) 43) the interaction between (+)-S-vorozole and the I-helix of cytochrome P450 19 (P450 aromatase) has been reported. In the present study we extended the "I-helix model" by incorporating the C-terminus of P450 aromatase. The crystal structures of P450 101 (P450 cam), 102 (P450 BM-3) and 108 (P450 terp) reveal that the C-terminus is structurally conserved and forms part of their respective substrate binding pocket. Furthermore, the present study is extended to the interaction between P450 aromatase and its natural substrate androstenedione and the non-steroidal inhibitors (-)-R-vorozole, (-)-S-fadrozole, R-liarozole and (-)-R-aminoglutethimide. It is found that (+)-S-vorozole, (-)-S-fadrozole and R-liarozole bind in a comparable way to P450 aromatase and interact with both the I-helix (Glu302 and Asp309) and C-terminus (Ser478 and His480). The weak activity of (-)-R-aminoglutethimide might be attributed to a lack of interaction with the C-terminus.     

Comparative molecular field analysis of non-steroidal aromatase inhibitors: an extended model for two different structural classes

Aromatase is a cytochrome P450 isozyme, whose inhibition is known to be therapeutically relevant in the treatment of the breast cancer. A comparative molecular field analysis (CoMFA) has been carried out on a series of non-steroidal aromatase inhibitors belonging to two different structural classes. One subset of compounds consists of fadrozole analogues and was studied in a previous work, from which a 'local' 3-D quantitative structure-activity relationship (QSAR) model for the inhibition of aromatase was obtained. In the present paper, that model is extended to include a second subset of compounds bearing a tetralone nucleus and acting at the same enzyme site with the same mechanism as the azoles. The critical alignment step has been solved by using two different steroidal inhibitors of aromatase as rigid templates, on which the non-steroidal compounds have been superimposed. The final 3-D QSAR models are discussed in terms of predictivity and some implications regarding the steric and electronic requirements of steroidal and non-steroidal inhibitors are pointed out.     

Potent peptide inhibitors of human hepatitis C virus NS3 protease are obtained by optimizing the cleavage products

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.     

Rapid reversed-phase high-performance liquid chromatography method for quantitation, at high pH, of the recombinant apolipoprotein A-IMilano in Escherichia coli fermentation broth

In an effort to develop potent agents for reducing the levels of the active estrogen, estradiol, we developed a new category of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 1 inhibitors. The compounds described possess a butyl methyl alkylamide side chain linked to the C6 position of estradiol by a thioether. With a series of epimeric mixtures, an optimal side-chain length of five methylene groups (between the amide group and steroid part) was first determined. Thereafter, both C6 epimers of optimized mixture were obtained after high-pressure liquid chromatography separation. 1H and 13C NMR experiments were performed to confirm the stereochemistry of each epimer. The 6beta-orientation of the side-chain was found to be crucial for enzymatic inhibition. Indeed, for the optimized side-chain length, the compound with a beta-orientation (5: N-butyl,N-methyl 7-(3',17'beta-dihydroxy-1',3',5'( 10')-estratriene-6'beta-yl)-7-thiaheptanamide) was 70-fold more potent than the 6alpha-analog. Compound 5 did not inactivate 17beta-HSD type 1, suggesting a reversible inhibitor. In addition, it was found to be a more potent inhibitor than the substrate estrone itself or a panel of three known inhibitors.     

Interaction of substrate uridyl 3',5'-adenosine with ribonuclease A: a molecular dynamics study

A wealth of information available from x-ray crystallographic structures of enzyme-ligand complexes makes it possible to study interactions at the molecular level. However, further investigation is needed when i) the binding of the natural substrate must be characterized, because ligands in the stable enzyme-ligand complexes are generally inhibitors or the analogs of substrate and transition state, and when ii) ligand binding is in part poorly characterized. We have investigated these aspects in the binding of substrate uridyl 3',5'-adenosine (UpA) to ribonuclease A (RNase A). Based on the systematically docked RNase A-UpA complex resulting from our previous study, we have undertaken a molecular dynamics simulation of the complex with solvent molecules. The molecular dynamics trajectories of this complex are analyzed to provide structural explanations for varied experimental observations on the ligand binding at the B2 subsite of ribonuclease A. The present study suggests that B2 subsite stabilization can be effected by different active site groups, depending on the substrate conformation. Thus when adenosine ribose pucker is O4'-endo, Gln69 and Glu111 form hydrogen-bonding contacts with adenine base, and when it is C2'-endo, Asn71 is the only amino acid residue in direct contact with this base. The latter observation is in support of previous mutagenesis and kinetics studies. Possible roles for the solvent molecules in the binding subsites are described. Furthermore, the substrate conformation is also examined along the simulation pathway to see if any conformer has the properties of a transition state. This study has also helped us to recognize that small but concerted changes in the conformation of the substrate can result in substrate geometry favorable for 2',3' cyclization. The identified geometry is suitable for intraligand proton transfer between 2'-hydroxyl and phosphate oxygen atom. The possibility of intraligand proton transfer as suggested previously and the mode of transfer before the formation of cyclic intermediate during transphosphorylation are discussed.     

Cleavage activity of hepatitis C virus serine proteinase

To study the character of the hepatitis C virus (HCV) encoding serine proteinase and to search for inhibitors, a practical in vitro assay system using the purified enzyme and synthetic peptide substrates was established. The enzyme used was expressed in Escherichia coli as a fusion form with protein tags and purified to apparent homogeneity by single-step affinity chromatography. The purified enzyme exhibited proteolytic activity with pH optima of around eight, and the addition of NS4A fragments increased the activity as well as the thermal stability of the enzyme. The activity was inhibited by EDTA and some divalent ions, i.e., copper and zinc, though calcium, magnesium, and manganese were stimulative both in the presence and absence of the NS4A fragment. None of the common protease inhibitors, including serine protease inhibitors, effectively inhibited the activity. Based on the kinetic parameters of the cleavage reaction of the synthetic 20 mer peptides corresponding to the three cleavage sites, NS4A/4B, NS4B/5A, and NS5A/5B, the peptide with the NS5A/5B junction was found to be the most efficient substrate. Analysis of the minimal peptide substrate of NS5A/5B indicated that 5 to 7 amino acids on both sides of the junction were required for efficient cleavage. These findings are expected to contribute to the search for a proteinase inhibitor.     

Single-strand breaks in oligodeoxyribonucleotides induced by fission neutrons and gamma radiation and measured by gel electrophoresis: protective effects of aminothiols

In order to approach the detailed structure-function relationships of aromatase, we studied the inhibitory and inactivatory potencies of several steroidal androstenedione analogues (1: 4-hydroxyandrostenedione, 2: 4-acetoxyandrostenedione and 3: 7 alpha-(4'-amino)phenylthio-4-androstene-3, 17-dione) and non-steroidal imidazole derivatives (4: ketoconazole, 5: miconazole and 6: fadrozole) on equine aromatase in placental microsomes, a well established mammalian model. Human placental microsomes and the purified enzyme from equine testis were also used to compare inhibition by 1 and 2. In equine microsomes, all compounds tested exhibited a competitive inhibition, with Ki values of 4.1, 26 and 1.8 nM for 1, 2 and 3, and of 2400, 1.4 and 4 nM for 4, 5, and 6, respectively. The Km for androstenedione, the substrate mainly used in these studies, was 1.8 +/- 0.13 nM. The three non-steroidal derivatives did not inactivate equine aromatase, but 1 and 2 acted as comparable inactivators to a much higher degree than 3. Compound 1 inhibited in a similar manner (89-94%) purified or equine and human microsomal aromatases, whereas 2 inhibited microsomal aromatase more efficiently in the horse than in man (92% and 33% inhibition, respectively). There was only a 40% inhibition with 2 on the purified equine enzyme, which is no more in the natural membrane environment. The comparisons between equine and human microsomal aromatases allow precise functional and structural differences to be observed with these enzymes.     

Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers

Microsomal glutathione transferase-1 (MGST-1) is an abundant protein that catalyzes the conjugation of electrophilic compounds with glutathione, as well as the reduction of lipid hydroperoxides. Here we report that leukotriene C4 is a potent inhibitor of MGST-1. Leukotriene C4 was found to be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for the N-ethylmaleimide activated enzyme. This is the first tight-binding inhibitor characterized for this enzyme. Leukotriene C4 was competitive with respect to glutathione and non-competitive toward the second substrate, CDNB. Analysis of stoichiometry supports binding of one molecule of inhibitor per homotrimer. Leukotrienes A4, D4, and E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues, which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078, and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3. 7, and 7.6 microM, respectively). In view of the partial structural similarity between MGST-1, leukotriene C4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC50 for BAYx1005 was 58 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent inhibitors of MGST-1 raises the possibility that MGST-1 influences the cellular processing of leukotrienes. These findings may also have implications for the effects and side-effects of drugs developed to manipulate leukotrienes.     

Design and synthesis of new potent C2-symmetric HIV-1 protease inhibitors. Use of L-mannaric acid as a peptidomimetic scaffold

A study on the use of derivatized carbohydrates as C2-symmetric HIV-1 protease inhibitors has been undertaken. L-Mannaric acid (6) was bis-O-benzylated at C-2 and C-5 and subsequently coupled with amino acids and amines to give C2-symmetric products based on C-terminal duplication. Potent HIV protease inhibitors, 28 Ki = 0.4 nM and 43 Ki = 0.2 nM, have been discovered, and two synthetic methodologies have been developed, one whereby these inhibitors can be prepared in just three chemical steps from commercially available materials. A remarkable increase in potency going from IC50 = 5000 nM (23) to IC50 = 15 nM (28) was observed upon exchanging -COOMe for -CONHMe in the inhibitor, resulting in the net addition of one hydrogen bond interaction between each of the two -NH- groups and the HIV protease backbone (Gly 48/148). The X-ray crystal structures of 43 and of 48 have been determined (Figures 5 and 6), revealing the binding mode of these inhibitors which will aid further design.     

The active site of Paracoccus denitrificans aromatic amino acid aminotransferase has contrary properties: flexibility and rigidity

Paracoccus denitrificans aromatic amino acid aminotransferase (EC 2. 6.1.57; pdAroAT) binds with a series of aliphatic monocarboxylates attached to the bulky hydrophobic groups. To analyze the properties of the active site in this enzyme, we determined the tertiary structures of pdAroAT complexed with nine different inhibitors. Comparison of these active site structures showed that the active site of pdAroAT consists of two parts with contrary properties: rigidity and flexibility. The regions that interact with the carboxylates and methylene chains of the inhibitors gave essentially the same structures among these complexes, exhibiting the rigid property, which would involve fixing the substrate at the proper orientation for efficient catalysis. The region that interacts with the terminal hydrophobic groups of the inhibitors gave versatile structures according to the structures of the terminal groups, showing that this region is structurally flexible. This is mainly achieved by the conformational versatility of the side chains of Asp15, Lys16, Asn142, Arg292, and Ser296. These residues formed in the active site hydrogen bond networks, which were adaptable for the structures of the terminal hydrophobic groups of the inhibitors, with a small deformation or partial destruction according to the shapes and sizes of the inhibitors. These observations illustrate how the flexibility and rigidity in the active site can be used for the substrate binding and recognition.     

Biochemistry and pharmacokinetics of potent non-steroidal cytochrome P450(17alpha) inhibitors

Two potent non-steroidal inhibitors (CB7645 and CB7661) of human cytochrome P450(17alpha) were tested for in vivo activity in WHT mice. There were no signs of toxicity, but there was no effect on the androgen-dependent organs. The pharmacokinetics and biochemistry of the compounds in mice were investigated. Following i.p. administration of 0.5 mmol/kg of CB7645 and CB7661, peak plasma levels of 13.4 and 3.4 microM, respectively, occurred after 2-4 h, both compounds were cleared rapidly (terminal half-lives 2.7 and 3.3 h, respectively) and neither was detectable at 24 h. CB7645 produced some decrease in plasma testosterone at 4 h, but this was not sustained. When tested in vitro against the WHT testicular enzyme, the CB7645 and CB7661 were competitive inhibitors with K(i) values of 10 and 13 nM, respectively. However, the K(m) for the substrate progesterone was lower at 4.3 nM. These data indicate that, for effective and continuous inhibition of the murine cytochrome P450(17alpha) enzyme, higher peak levels of the compounds would be required, and these levels would need to be maintained throughout the treatment period.     

The effects of temperature and some inhibitors on the carnitine exchange system of heart mitochondria

1. 11-Trimethylamino-undecanoyl-L-carnitine (Ki = 56 muM) and 6-trimethylaminohexanoyl-L-carnitine (Ki = 1.3 mM) are competitive inhibitors of the ox heart mitochondrial carnitine exchange system. The latter is itself a poor substrate, with a V about 0.7% of that for carnitine. These compounds have been used as 'stop inhibitors' in a study of the effect of temperature on the carnitine exchange. 2. The carnitineout in equilibrium carnitinein exchange has a temperature coefficient (Q10) of 8.5 and an activation energy of 176 kJ/mol which is constant from 0 degrees - 18 degrees C. The Km for L-carnitine (5.3 mM) is also constant over this temperature range. 3. The slow efflux (leak) of mitochondrial L-[14C]carnitine in the absence of external substrate has a similar temperature dependence and is also inhibited by 11-trimethylamino-undecanoyl-L-carnitine. It is probably mediated by the same system as the very much faster exchange with external carnitine.     

Drug treatment of prostatic carcinoma

The basis for the medical treatment of prostate cancer is inhibition of the influence of testosterone on the prostate. Surgical castration is in 1997 still the gold standard; it reduces the testosterone level by 90% and the level of dihydrotestosterone (the active metabolite) by 60%. In the eighties luteinising hormone releasing hormone (LH-RH) analogues were introduced to avoid the psychological burden of castration. After an initial stimulation (the flare-up) testosterone decreases to castrate level within 3 weeks. Recently (non-steroidal) anti-androgens, competitive inhibitors of dihydrotestosterone on receptor level were introduced. There are also drugs which inhibit the conversion of testosterone to dihydrotestosterone: 5 alpha-reductase inhibitors. Non-steroidal anti-androgens and 5 alpha-reductase inhibitors do not decrease the testosterone level and therefore cause less loss of libido and energy than castration. Combination of (chemical) castration and anti-androgens is called maximum androgen blockade. This treatment has limited additional value in proportion to the increase in side effects and costs. A new form of treatment is intermittent androgen blockade. With this strategy growth of hormone-insensitive cells in the prostate, which is considered the main determinant of the poor prognosis, might be delayed with reduction of side effects and costs. The role of imidazoles is still investigated; the role of cytotoxic drugs is mainly palliative.     

Product inhibition of the hepatitis C virus NS3 protease

The nonstructural protein NS3 of the hepatitis C virus (HCV) harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. Its inhibition is presently regarded as a promising strategy for coping with the disease caused by HCV. In this work, we show that the NS3 protease undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B cleavage sites, whereas no inhibition is observed with a cleavage product of the intramolecular NS3-NS4A junction. The Ki values of the hexamer inhibitory products [Ki(NS4A) = 0.6 microM, Ki(NS5A) = 1.4 microM, and Ki(NS4B) = 180 microM] are lower than the Km values of the respective substrate peptides [Km(NS4A-NS4B) = 10 microM, Km(NS5A-NS5B) = 3.8 microM, and Km(NS4B-NS5A) > 1000 microM]. Mutagenesis experiments have identified Lys136 as an important determinant for product binding. The phenomenon of product inhibition can be exploited to optimize peptide inhibitors of NS3 protease activity that may be useful in drug development.     

Preferential utilization of ketone bodies for the synthesis of myelin cholesterol in vivo

1. The distribution of radioactivity among lipid classes of myelin and other subcellular brain fractions of young rats (18-21 days) was determined after in vivo injection of (3-(14)C-labelled ketone bodies, [U-(14)C] glucose or [2-(14)C] glucose. 2. The incorporation ratios (sterol/fatty acids) were 0.67, 1.48, 0.25, 0.62 and 0.54 for whole brain, myelin, mitochondria, microsomes and synaptosomes, respectively, with (3-(14)C)-labelled ketone bodies as substrate and 0.37, 0.89, 0.19, 0.34 and 0.29 with [U-(14)C] glucose as substrate. These data show that, both in whole brain and in subcellular brain fractions, acetyl groups derived from ketone bodies are used for sterol synthesis to a large extent than acetyl groups originating from glucose. 3. The specific radioactivity of cholesterol is much higher in myelin than in whole brain or in the other brain fractions, particularly after administration of labelled ketone bodies as substrate. 4. The incorporation patterns of acetoacetate and D-3-hydroxybutyrate were very similar, indicating that both ketone bodies contribute acetyl groups for lipid synthesis via the same metabolic route. 5. Our data suggest that a direct metabolic path from ketone bodies towards cholesterol exists - possibly via acetoacetyl-CoA formation in the cytosol of brain cells - and that this process is most active in oligodendrocytes.     

Chemo-enzymatic synthesis of galactosylmaltooligosaccharidonolactone as a substrate analogue inhibitor for mammalian alpha-amylase

We performed chemo-enzymatic transformation of maltooligosaccharides into both end-modified oligosaccharidonolactones of potential use as substrate analogue inhibitors for mammalian alpha-amylases. Enzymatic modification of the non-reducing end glucosyl residue of the maltooligosaccharide was first performed by transglycosylation with beta-D-galactosidase from Bacillus circulans. When maltotriose and maltotetraose were the acceptors, the enzyme regioselectively synthesized 4(3)-O-beta-D-galactosyl maltotriose (LG3) and 4(4)-O-beta-D-galactosyl maltotetraose (LG4) from lactose as a donor. LG4 was further selectively hydrolyzed with a specific alpha-amylase to afford 4(2)-O-beta-D-galactosyl maltose (LG2). The anomer hydroxyl groups of LG2 and LG3 were chemically oxidized to give the corresponding lactones, 4(2)-O-beta-D-galactosyl maltobionolactone (LG2O) and 4(3)-O-beta-D-galactosyl maltotrionolactone (LG3O), respectively. LG2O and LG3O, which are competitive inhibitors for mammalian alpha-amylases, exhibited Ki values of the order of 2.8-18.0 microM, with p-nitrophenyl alpha-maltopentaoside (G5P) as the substrate. On 1H-NMR analysis, these oligosaccharidonolactones were shown to be transformed into the corresponding aldonic acid forms with time in an aqueous solution. In this case, the lactone form was essential for the occurrence of the alpha-amylase inhibitor.     

End-plate voltage-gated sodium channels are lost in clinical and experimental myasthenia gravis

Although the signaling pathways leading to hydrogen peroxide (H2O2)-induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. We have recently demonstrated that thrombin-induced actin reorganization in bovine pulmonary artery endothelial cells (BPAEC) requires activation of both myosin light chain kinase (MLCK) and protein kinase C (PKC). Therefore, the present study was designed to investigate the effects of H2O2 on actin reorganization in BPAEC. H2O2 initiated sustained recruitment of actin to the cytoskeleton and transient myosin recruitment in a time- and concentration-dependent manner. The H2O2-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP, but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 and calphostin C. H2O2 also caused actin filament rearrangement in BPAEC with disruption of the dense peripheral bands and formation of stress fibers. These alterations occurred prior to actin translocation to the cytoskeleton and are prevented by inhibition of either MLCK or PKC. High concentrations of H2O2 transiently attenuated PKC activity but slightly increased the phosphorylation of the prominent PKC substrate and actin-binding protein, myristoylated alanine-rich C kinase substrate (MARCKS), by 5 min. However, MARCKS phosphorylation was reduced to below basal levels by 30 min. On the other hand, H2O2 induced a time- and dose-dependent phosphorylation of myosin light chains which was eliminated by both MLCK and PKC inhibitors. These data suggest that MLCK contributes to H2O2-induced myosin light chain phosphorylation and actin rearrangement and that PKC may play a permissive role. Neither of these enzymes appears to be involved in the H2O2-induced recruitment of actin to the cytoskeleton.     

Effect of protein kinase inhibitors on activity of mammalian small heat-shock protein (HSP25) kinase

The aim of this study was to investigate different protein kinase inhibitors (secondary metabolite-derived substances, synthetic compounds, and substrate-based peptides) for their potency to inhibit the mammalian small heat shock protein (HSP25) kinase (E.C. 2.7.1.37) isolated from Ehrlich ascites tumor cells. Among the secondary metabolite-derived inhibitors (staurosporine, K-252a, K-252b, KT5926, KT5720, erbstatin analog, and quercetin) and synthetic compounds (H-9, H-89, HA 1004, KN-62, ML-7, tyrphostin A25, and tyrphostin B42), KT5926, staurosporine, and K-252a inhibited HSP25 kinase most efficiently. Kinetic analysis revealed that inhibition by staurosporine (Ki = 32.4 nM) and K-252a (Ki = 13.7 nM) was competitive with ATP. Inhibition by KT5926 was competitive with the substrate peptide KKKALNRQLSVAA (Ki = 27.2 nM) and noncompetitive with respect to ATP (Ki = 38.8 nM). In comparison with other protein kinases, HSP25 kinase was relatively resistant to most of the inhibitors. KT5926 was the only tested inhibitor with certain preference for HSP25 kinase when compared with protein kinases A, C, and G. Among the tested substrate-based peptides, we identified one peptide (KKKALNRQLGVAA), which preferentially inhibited HSP25 kinase in comparison with protein kinases A and C and mitogen-activated protein kinase. This peptide inhibited HSP25 kinase competitively with the substrate peptide (Ki = 8.1 microM) and noncompetitively with ATP (Ki = 134 microM). A peptide (SRVLKEDKERWEDVK) derived from the putative autoinhibitory domain of the closely related human mitogen-activated protein kinase-activated protein kinase-2 did not inhibit HSP25 kinase activity, suggesting the existence of several species of HSP25 kinases. Furthermore, the data identified structural requirements for inhibitors of HSP25-kinase.     

Ras-GRF, the activator of Ras, is expressed preferentially in mature neurons of the central nervous system

Neprilysin is a neutral peptidase that cleaves small peptide substrates on the amino-side of hydrophobic amino acid residues. In the present study, we have used inhibition of non-mutated and mutated enzymes with dipeptide inhibitors and hydrolysis of the substrate [Leu5, Arg6]enkephalin in order to evaluate the contribution of the S2' subsite to substrate and inhibitor binding. Our results suggest that (1) Arg-102 and Asn-542 provide major contributions to the interaction of the enzyme with the P2' residue of the substrate, (2) the S2' subsite is vast and can accommodate bulky side chains, and (3) Arg-102 restricts access to the S2' subsite to some side chains such as arginine.