Structure-based rational design of peptide hydroxamic acid inhibitors to target tumor necrosis factor-α converting enzyme as potential therapeutics for hepatitis

The human tumor necrosis factor-α converting enzyme (TACE) has recently been raised as a new and promising therapeutic target of hepatitis and other inflammatory diseases. Here, we reported a successful application of the solved crystal structure of TACE complex with a peptide-like ligand INN for rational design of novel peptide hydroxamic acid inhibitors with high potency and selectivity to target and inhibit TACE. First, the intermolecular interactions between TACE catalytic domain and INN were characterized through an integrated bioinformatics approach, with which the key substructures of INN that dominate ligand binding were identified. Subsequently, the INN molecular structure was simplified to a chemical sketch of peptide hydroxamic acid compound, which can be regarded as a linear tripeptide capped by a N-terminal carboxybenzyl group (chemically protective group) and a C-terminal hydroxamate moiety (coordinated to the Zn²⁺ at TACE active site). Based on the sketch, a virtual combinatorial library containing 180 peptide hydroxamic acids was generated, from which seven samples were identified as promising candidates by using a knowledge-based protein–peptide affinity predictor and were then tested in vitro with a standard TACE activity assay protocol. Consequently, three designed peptide hydroxamic acids, i.e. Cbz-Pro-Ile-Gln-hydroxamic acid, Cbz-Leu-Ile-Val-hydroxamic acid and Cbz-Phe-Val-Met-hydroxamic acid, exhibited moderate or high inhibitory activity against TACE, with inhibition constants K_i of 36?±?5, 510?±?46 and 320?±?26?nM, respectively. We also examined the structural basis and non-bonded profile of TACE interaction with a designed peptide hydroxamic acid inhibitor, and found that the inhibitor ligand is tightly buried in the active pocket of TACE, forming a number of hydrogen bonds, hydrophobic forces and van der Waals contacts at the interaction interface, conferring both stability and specificity for TACE–inhibitor complex architecture.     

Inhibition of acetylcholinesterase by dibenamine and dibenzyline

Dibenamine and dibenzyline are irreversible inhibitors of acetylcholinesterase (AChE). Kinetic studies show that at pH 9.5 a fast reaction occurs between a group on the enzyme with pK_a 9.1 and the ethyleniminium ion derived from the inhibitor. Either the ε-amino-group of a lysine residue is alkylated or else a lysine residue catalyses the alkylation of a non-ionisable group (e.g. hydroxyl). At pH 6.5 there is a slow reaction between a carboxyl anion on the enzyme and the ethyleniminium ion. Studies of the alkylation reactions in the presence of the reversible competitive inhibitor of the enzyme, tetramethylammonium ion, show that alkylation occurs at some distance from the anionic site and probably on the borders of the active site.     

N-myristoylation of a peptide substrate for Src converts it into an apparent slow-binding bisubstrate-type inhibitor

Abstract: The conversion of a peptide substrate to a potent inhibitor by chemical modification is a promising approach in the development of inhibitors for protein tyrosine kinases. N-acylation of the synthetic peptide substrate NH₂-Glu-Phe-Leu-Tyr-Gly-Val-Phe-Asp-CONH₂ (EFLYGVFD) resulted in synergistic inhibition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was dependent upon the peptide sequence and the length of the acyl chain. The minimum length of the fatty acyl chain to synergistically inhibit Src was a lauryl (C₁₁H₂₃CO) group. N-myristoylated EFLYGVFD (myr-EFLYGVFD) inhibited the phosphorylation of poly E₄Y by Src with an apparent K_i of 3 µm , whereas EFLYGVFD and myristic acid inhibited with K_i values of 260 and 35 µm , respectively. The nonacylated EFLYGVFD was a substrate for Src with K_m and V_max values of 100 µm and 400 nmol/min/mg protein, respectively. However, upon myristoylation, the peptide was no longer a substrate for Src. Both the acylated and non-acylated peptides were competitive inhibitors against the substrate poly E₄Y. The non-acylated free peptide showed mixed inhibition against ATP while the myristoylated peptide was competitive against ATP. Myristic acid was uncompetitive against poly E₄Y and competitive against ATP. Further analysis indicated that the myristoylated peptide acted as a reversible slow-binding inhibitor with two binding sites on Src. The myristoylated 8-mer peptide was reduced in size to a myristoylated 3-mer without losing the affinity or charactersitics of a bisubstrate-type inhibitor. The conversion of a classical reversible inhibitor to a reversible slow-binding multisubstrate analogue has improved the potency of inhibition by the peptide.     

Purification and characterization of Locusta migratoria chymotrypsin

A chymotrypsin-like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion-exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS-PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24 000, determined by SDS-PAGE and on a Sephadex G-75 calibrated column. It has an isoelectric point of 10.1 and contains 0-1 half cystine residues. Sequence analysis of the first 20 N-terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5–9.0. The K_m and k_cat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, were similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK, indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double-headed, chymotrypsin-trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI), which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide-chloromethylketones point towards the existence of an extended binding site.     

Syntheses of argininal semicarbazone containing peptides and their applications in the affinity chromatography of serine proteinases†

Eight argininal semicarbazone containing peptides prepared by liquid phase synthesis were all found to be reversible inhibitors of model serine proteinases including trypsin and plasma kallikrein (PK). Among the peptides tested, those having a Lys residue at position P₂ displayed the maximum binding potency towards PK. One of the peptides, Leu-enkephalin-argininal semicarbazone, a comparatively weak inhibitor, was chosen in order to develop an affinity-based purification protocol for PK. The affinity column was prepared by covalent attachment of the NH₂ -terminal moiety of the peptidyl semicarbazone to a solid-phase matrix bearing a spacer group. For efficient binding of PK, it was found necessary to optimize parameters like the concentration of inhibitor linked to the solid matrix, the ionic strength of the buffer used, the temperature and the pH. The majority of the bound enzyme could be recovered following elution with guanidine hydrochloride or benzamidine hydrochloride in a high salt buffer at pH 6.0. The usefulness of the affinity procedure towards the purification of other serine proteinases is also discussed.     

Design and structure of symmetry-based inhibitors of HIV-1 protease

Summary The concept of active-site symmetry along with structural and mechanistic considerations of aspartic proteases has been used to design novel,C ₂-symmetry-based inhibitors of HIV PR. A comparative molecular-modeling strategy was used in the design of a pseudo-C ₂-symmetric diaminoalcohol series and later a diastereomeric series ofC ₂-symmetric diaminodiols. The structure of the pseudosymmetric alcohol A-74704, complexed with HIV PR, confirmed the proposed symmetric mode of binding, and also proved useful in subsequent efforts to improve the solubility of the more potent diol series. The crystal-structure analysis of a pseudosymmetricR,S-diol provided insights into the relative contributions of the two hydroxyl groups to the stability of the complex. A comparison of the binding modes for symmetric and asymmetric, substrate-based inhibitors indicates that the influence of symmetry on binding is mainly expressed at the protein level, since the overall subsite symmetry is maintained even with highly asymmetric inhibitors. Thus, while the incorporation ofC ₂ symmetry does not necessarily lead to compounds with improved potency over asymmetric compounds, this strategy has led to the design of structurally novel and perhaps pharmacologically desirable inhibitors. The growing number of examples of symmetry-based inhibitors is evidence that symmetry has become a useful paradigm for HIV protease inhibitor design and that structure-based approaches to inhibitor design can contribute in unique and critical ways to the conceptualization of medicinal chemistry strategies that can lead to useful clinical candidates for AIDS, cancer and other diseases.     

Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung

A sensitive, fixed-time, spectrophotometric assay for angiotensin-converting enzyme measures the rate of production of hippuric acid from hippuryl-L-histidyl-L-leucine (HHL). The angiotensin-converting enzyme from rabbit lung acetone powder extract, when assayed by this method, is optimally active at pH 8.1 to 8.3 at a chloride ion concentration of 300 mM and an HHL concentration of 5–10 mM; the K_m for HHL is 2–6 mM. The enzyme was inhibited by metal-chelating agents, heavy metal salts and certain peptides. The most effective inhibitors were EDTA; CdBr₂; angiotensin II; bradykinin; and a pentapeptide, L-pyroglutamyl-L-lysyl-L-tryptophyl-L-alanyl-L-proline, a component of Bothrops jararaca venom. Enzyme inhibited by 0.1 mM EDTA was completely reactivated after removal of EDTA by dialysis but, after prolonged dialysis of the enzyme against 1 mM EDTA, reactivation could only be achieved by addition of metal ions: MnCI₂ (40%), ZnCl₂ (100%) or Co_(NO3)2 (160%). The angiotensin-converting enzyme of rabbit lung is a stable, chloride ion-activated metalloenzyme, similar to both the angiotensin-converting enzyme and kininase II of plasma.     

A second N-acylethanolamine hydrolase in mammalian tissues

It is widely accepted that fatty acid amide hydrolase (FAAH) plays a central role in the hydrolysis of anandamide. However, we found a second N-acylethanolamine hydrolase in animal tissues which hydrolyzed anandamide at acidic pH. This “acid amidase” was first detected with the particulate fraction of human megakaryoblastic CMK cells, and was solubilized by freezing and thawing without detergent. The enzyme was distinguishable from FAAH in terms of (1) the optimal activity at pH 5, (2) stimulation by dithiothreitol, (3) low sensitivity to two FAAH inhibitors (methyl arachidonyl fluorophosphonate and phenylmethylsulfonyl fluoride), and (4) high content in lung, spleen and macrophages of rat. The acid amidase purified from rat lung was the most active with N-palmitoylethanolamine among various long-chain N-acylethanolamines. To develop specific inhibitors for this enzyme, we screened various analogues of N-palmitoylethanolamine. Among the tested compounds, N-cyclohexanecarbonylpentadecylamine was the most potent inhibitor which does-dependently inhibited the enzyme with an IC₅₀ value of 4.5 μM without inhibiting FAAH at concentrations up to 100 μM. The inhibitor was a useful tool to distinguish the acid amidase from FAAH with rat basophilic leukemia (RBL-1) cells that express both the enzymes.     

Effects of indomethacin, 5,8,11,14-eicosatetraynoic acid, and p-bromophenacyl bromide on lysosomal enzyme release and superoxide anion generation by human polymorphonuclear leukocytes

Preincubation of cytochalasin B-treated, human polymorphonuclear leukocytes (PMN) with indomethacin (a cyclo-oxygenase inhibitor), 5,8,11,14-eicosatetraynoic acid (ETYA) (a lipoxygenase/cyclo-oxygenase inhibitor), or p-bromophenacyi bromide (BPB) (a phospholipase A₂ inhibitor) resulted in dose-dependent inhibition of lysosomal enzyme release elicited by the chemotactic peptide N-formylmethionylleucylphenylalanine (FMLP); 50 per cent inhibition was seen at approximately 50, 12, 8 μM respectively. BPB also inhibited Superoxide anion generation. The effects of indomethacin and ETYA were dependent upon the type of stimulus presented to the cells. Lysosomal enzyme release stimulated by zymosan-treated serum and serum-treated zymosan was relatively unaffected by these two inhibitors. Indomethacin and ETYA did not appear to exert their effects by specific inhibition of prostaglandin and thromboxane synthesis; the inhibition offered by both agents was reversible, and aspirin had no similar inhibitory capacity. Our results indicate not only that indomethacin may exert effects independent of its inhibition of the cyclo-oxygenase pathway but also that products formed via phospholipase and lipoxygenase may be mediators of lysosomal enzyme release and superoxide anion generation.     

Design of potent substrate-analogue inhibitors of canine renin

Through a systematic study of structure-activity relationships, we designed potent renin inhibitors for use in dog models. In assays against dog plasma renin at neutral pH, we found that, as in previous studies of rat renin inhibitors, the structure at the P2 position appears to be important for potency. The substitution of Val for His at this position increases potency by one order of magnitude. At the P3 position, potency appears to depend on a hydrophobic side chain that does not necessarily have to be aromatic. Our results also support the approach of optimizing potency in a renin inhibitor by introducing a moiety that promotes aqueous solubility (an amino group) at the C-terminus of the substrate analogue. In the design of potent dog plasma renin inhibitors, the influence of the transition-state residue 4(S)-amino-3(S)-hydroxy-5-cyclohexylpentanoic acid (ACHPA) - commonly used as a substitute for the scissile-bond dipeptide to boost potency - is not obvious, and appears to be sequence dependent. The canine renin inhibitor Ac-paF-Pro-Phe-Val-statine-Leu-Phe-paF-NH₂ (compound 15; IC₅₀ of 1.7 nM against dog plasma renin at pH 7.4; statine, 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid; paF, para-aminophenylalanine) had a potent hypotensive effect when infused intravenously into conscious, sodium-depleted, normotensive dogs. Also, compound 15 concurrently inhibited plasma renin activity and had a profound diuretic effect.     

AFFINITY CHROMATOGRAPHY OF SEVERAL PROTEOLYTIC ENZYMES ON CARBOBENZOXY-D-PHENYLALANYL-TRIETHYLENETETRAMINE-SEPHAROSE*

Carbobenzoxy-D-phenylalanyl-triethylenetetramine-Sepharose (Z-D-Phe-T-Sepharose) was found to be an effective affinity adsorbent for several proteolytic enzymes which preferentially catalyze the hydrolytic cleavage of peptide bonds involving amino or carboxyl groups of hydrophobic and bulky amino acid residues. α-Chymotrypsin, subtilisin and neutral metalloendopeptidase were adsorbed at pH 5–7, while pepsin and Rhizopus niveus acid protease showed affinity to the adsorbent at pH 3.5–5.0. α-Chymotrypsin was recovered by elution with 0.1 M acetic acid and neutral subtilopeptidase (B. subtilis neutral protease) was eluted with 0.5 M NaCl at pH 9.0. Subtilisin and thermolysin were found in the eluates with 1.5 M guanidine-HCl, 1 M p-toluenesulfonate or 30% ethylene glycol at pH 7. Rhizopus acid protease was eluted with 0.5 M acetic acid and pepsin with 1 M guanidine-HCl or p-toluenesulfonate at pH 3.5. The chromatographic purifications by this method of highly purified or crystallized enzyme preparations commercially available resulted in 1.3–1.5-fold increase in their specific activities. Inactivation of these proteolytic enzymes by chemical modification, and formation of enzyme-inhibitor complex led to loss of the binding ability to the adsorbent. On the other hand, the zinc-free apoenzymes of neutral subtilopeptidase and thermolysin were adsorbed onto and eluted from the adsorbent column under the same conditions as those for the native enzymes, suggesting that zinc is not essential for the binding of the substrate to these metalloproteases.     

Effect of the addition of a beta-blocker on left ventricular remodeling and prognosis in patients with dilated cardiomyopathy treated with angiotensin-converting enzyme inhibitor

To examine the effect of the addition of a beta-blocker in the treatment of chronic heart failure due to dilated cardiomyopathy, we compared the change of left ventricular remodeling and the prognosis between patients treated with angiotensin-converting enzyme inhibitors and patients who had beta-blockers added to angiotensin-converting enzyme inhibitors. Fifty-seven patients were treated with an angiotensin-converting enzyme inhibitor in addition to combination therapy with furosemide, spironolactone and digoxin. In 60 patients, a beta-blocker was administered in addition to combination therapy with furosemide, spironolactone, digoxin and an angiotensin-converting enzyme inhibitor. Changes of left ventricular dimensions at end-diastole and end-systole, fractional shortening, cardiac events and death were examined during the follow-up periods. The mean follow-up periods were 4.9 +/- 4.1 years in the angiotensin-converting enzyme inhibitor group and 3.9 +/- 2.5 years in the beta-blocker group, respectively. Baseline hemodynamic characteristics showed no significant differences between the two groups. After the treatment, the heart rate significantly decreased in both groups and the systolic blood pressure increased in the beta-blocker group. Both left ventricular dimensions at end-diastole and end-systole significantly decreased in both groups. Fractional shortening increased from 17.0 +/- 7.6 to 19.8 +/- 8.9% (p = 0.017) in the angiotensin-converting enzyme inhibitor group and from 16.6 +/- 7.2 to 24.7 +/- 8.0% (p < 0.0001) in the beta-blocker group, respectively. Changes of left ventricular dimensions at end-diastole and at end-systole, and fractional shortening were all greater in the beta-blocker group than in the angiotensin-converting enzyme inhibitor group. The event-free rate and the cumulative survival rate during the follow-up periods were markedly better in the beta-blocker group than in the angiotensin-converting enzyme inhibitor group (p = 0.0019 and p = 0.0099, respectively). These results indicate that the suppression of left ventricular remodeling and the improvement of prognosis in patients with dilated cardiomyopathy are markedly stronger in the beta-blocker group than in the angiotensin-converting enzyme inhibitor group. Thus, beta-blocker should be added to patients with dilated cardiomyopathy treated with an angiotensin-converting enzyme inhibitor.     

Anti-inflammatory property of n-hexadecanoic acid: structural evidence and kinetic assessment

Ester bond hydrolysis of membrane phospholipids by Phospholipase A₂ and consequent release of fatty acids are the initiating steps of inflammation. It is proposed in this study that the inhibition of phospholipase A₂ is one of the ways to control inflammation. Investigations are carried out to identify the mode of inhibition of phospholipase A₂ by the n‐hexadecanoic acid. It may help in designing of specific inhibitors of phospholipase A₂ as anti‐inflammatory agents. The enzyme kinetics study proved that n‐hexadecanoic acid inhibits phospholipase A₂ in a competitive manner. It was identified from the crystal structure at 2.5 Å resolution that the position of n‐hexadecanoic acid is in the active site of the phospholipase A₂. The binding constant and binding energy have also been calculated using Isothermal Titration Calorimetry. Also, the binding energy of n‐hexadecanoic acid to phospholipase A₂ was calculated by in silico method and compared with known inhibitors. It may be concluded from the structural and kinetics studies that the fatty acid, n‐hexadecanoic acid, is an inhibitor of phospholipase A₂, hence, an anti‐inflammatory compound. The inferences from the present study validate the rigorous use of medicated oils rich in n‐hexadecanoic acid for the treatment of rheumatic symptoms in the traditional medical system of India, Ayurveda.     

Crystal structure of the complex of group I PLA2 with a group II-specific peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) at 2.6 Å resolution

Phospholipases A₂s (PLA₂s) are widely distributed in mammals and snake venoms. They catalyze the production of arachidonic acid from membrane phospholipids leading to the bioynthesis of pro-inflammatory eicosanoids. A peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) was designed and synthesized as a specific inhibitor of PLA₂. It was shown earlier that the peptide bound to group II PLA₂ specifically and had a dissociation constant (K_d) of 8.8 × 10^{? 9} M. In the present studies for the binding of LAIYS with a group I PLA₂ from Naja naja sagittifera using surface plasmon resonance the dissociation constant was found to be 4.5 × 10^{? 5} M which is considerably lower than the value found for the group II PLA₂. In order to determine the details of binding at the molecular level, a group I PLA₂ from the venom of Naja naja sagittifera was crystallized with peptide LAIYS. The crystal structure showed the presence of LAIYS at the substrate-binding site but has fewer interactions than those observed with group II PLA₂ from Daboia russelli pulchella. The observed difference in the binding affinity is caused primarily due to poor fitting of the peptide LAIYS in the binding site of group I PLA₂. Apparently, the location of Trp 19 in group I PLA₂ is not favourable for the binding of LAIYS. The two complexes also differ drastically in the formation of intermolecular interactions. In the present structure, the side chain of Ser (P) interacts with His 48 and Asp 49 while in the complex with group II PLA₂ it was Tyr (P) OH that formed the corresponding interactions. Tyr (P) in group I PLA₂ is the main contributor of the hydrophobic interactions whereas in the complex of LAIYS with group II PLA₂ it was the peptide segment Leu-Ala-Ile that produced the bulk of hydrophobic forces. The structures further showed that the peptide LAIYS was fully inside the substrate-binding region of the group II PLA₂ while a significant portion of the peptide LAIYS was hanging outside the surface of the group I PLA₂. The buried area in the complex with group II PLA₂ was 811 Ų whereas, the corresponding area in group I PLA₂ was 449 Ų. This shows that the peptide LAIYS is very compatible with the substrate-binding site of group II PLA₂ and rather poorly fits into the substrate-binding site of group I PLA₂. This indicates that a highly specific ligand for one form of PLA₂ may be a poor partner for another form of enzyme.     

Inhibition of tyrosinase by 4H‐chromene analogs: Synthesis,kinetic studies,and computational analysis

Inhibition of mushroom tyrosinase was observed with synthetic dihydropyrano[3,2‐b]chromenediones. Among them, DHPC04 displayed the most potent tyrosinase inhibitory activity with a K_i value of 4 μm , comparable to the reference standard inhibitor kojic acid. A kinetic study suggested that these synthetic heterocyclic compounds behave as competitive inhibitors for the L‐DOPA binding site of the enzyme. Furthermore, molecular modeling provided important insight into the mechanism of binding interactions with the tyrosinase copper active site.     

Effects of pH and Dose on Nasal Absorption of Scopolamine Hydrobromide in Human Subjects

Purpose. The present study was conducted to evaluate theeffects of formulation pH and dose on nasal absorption of scopolaminehydrobromide, the single most effective drug available for the prevention ofnausea and vomiting induced by motion sickness. Methods. Human subjects received scopolamine nasally at adose of 0.2 mg/0.05 mL or 0.4 mg/0.10 mL, blood samples were collected atdifferent time points, and plasma scopolamine concentrations were determinedby LC-MS/MS. Results. Following administration of a 0.2 mg dose, theaverage C_max values were found to be 262 ± 118, 419± 161, and 488 ± 331 pg/mL for pH 4.0, 7.0, and 9.0formulations, respectively. At the 0.4 mg dose the average C_maxvalues were found to be 503 ± 199, 933 ± 449, and 1,308± 473 pg/mL for pH 4.0, 7.0, and 9.0 formulations, respectively. At a0.2 mg dose, the AUC values were found to be 23,208 ± 6,824, 29,145± 9,225, and 25,721 ± 5,294 pg.min/mL for formulation pH 4.0,7.0, and 9.0, respectively. At a 0.4 mg dose, the average AUC value wasfound to be high for pH 9.0 formulation (70,740 ± 29,381 pg.min/mL)as compared to those of pH 4.0 (59,573 ± 13,700 pg.min/mL) and pH 7.0(55,298 ± 17,305 pg.min/mL) formulations. Both the C_maxand AUC values were almost doubled with doubling the dose. On the otherhand, the average T_max values decreased linearly with a decreasein formulation pH at both doses. For example, at a 0.4 mg dose, the averageT_max values were 26.7 ± 5.8, 15.0 ± 10.0, and 8.8± 2.5 minutes at formulation pH 4.0, 7.0, and 9.0, respectively. Conclusions. Nasal absorption of scopolamine hydrobromidein human subjects increased substantially with increases in formulation pHand dose.     

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B2 and M1 muscarinic acetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (<EKWAP), a member of this structurally related peptide family, was essential for the development of captopril, the first site-directed ACE inhibitor used for the treatment of human hypertension. Nowadays, more Bj-PROs have been identified with higher ACE inhibition potency compared to Bj-PRO-5a. However, despite its modest inhibitory effect of ACE inhibition, Bj-PRO-5a reveals strong bradykinin-potentiating activity, suggesting the participation of other mechanisms for this peptide. In the present study, we have shown that Bj-PRO-5a induced nitric oxide (NO) production depended on muscarinic acetylcholine receptor M1 subtype (mAchR-M1) and bradykinin B₂ receptor activation, as measured by a chemiluminescence assay using a NO analyzer. Intravital microscopy based on transillumination of mice cremaster muscle also showed that both bradykinin B₂ receptor and mAchR-M1 contributed to the vasodilatation induced by Bj-PRO-5a. Moreover, Bj-PRO-5a-mediated vasodilatation was completely blocked in the presence of a NO synthase inhibitor. The importance of this work lies in the definition of novel targets for Bj-PRO-5a in addition to ACE, the structural model for captopril development.     

Carrier-mediated intestinal absorption of valacyclovir,the L-valyl ester prodrug of acyclovir. 1. Interactions with peptides,organic anions and organic cations in rats

The mechanism of intestinal transport of valacyclovir (VACV), the L-valyl ester prodrug of acyclovir, was investigated in rats using an in situ intestinal perfusion technique. VACV demonstrates an oral bioavailability that is three to five time greater than acyclovir, concentration dependent, and saturable in humans. Homogenate and perfused buffer stability results demonstrated that VACV was increasingly unstable with increasing pH. VACV was converted to ACV in a concentration dependent manner during a single pass through the intestinal segment. Perfusions were performed at 37°C, pH 6.5, and under iso-osmotic conditions (290±10 mOsm L⁻¹). Intestinal outlet concentrations were corrected for VACV that was converted to ACV during the perfusion. The effective dimensionless intestinal permeability (P_e*) of VACV was concentration dependent, saturable (intrinsic K_m = 1.2±0.7 mM), and significantly reduced (p <0.05) in the presence of peptide analogues (amoxicillin, ampicillin, cefadroxil, and cephradine), by the organic anion, p -amino hippuric acid and by the organic cation quinine. VACV transport was not inhibited by classical nucleoside competitive substrates or inhibitors or by valine. These results suggest that H⁺–oligopeptide, H⁺–organic cation, and organic anion transporters are involved in the small-intestinal uptake of VACV. The permeability of VACV in the colon was very low, indicating that VACV is predominantly absorbed from the small intestine. VACV P_e* was not altered in the presence of glucose-induced convective fluid flow, suggesting that carrier-mediated, transcellular uptake is the predominant absorption pathway of VACV in rat small intestine. Based on these results, the oral bioavailability of VACV appears to be significantly influenced by the preabsorptive conversion of VACV to the poorly absorbed ACV, by the involvement of multiple transporters in VACV small-intestinal uptake, and by the low permeability of VACV in the colon. © 1998 John Wiley & Sons, Ltd.     

Pepsin as a catalyst of peptide synthesis Enzyme co-precipitation with emerging peptide products

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH₃ formation, an equilibrium yield was attained at 1:3. 10⁵ enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.     

Impact of curcumin on the pharmacokinetics of rosuvastatin in rats and dogs based on the conjugated metabolites

1. Plasma concentrations of curcumin-O-glucuronide (COG) and curcumin-O-sulfate (COS) significantly increased after Sprague-Dawley rats dealt with the Oatp inhibitor rifampicin, with the C_max ascending 2.9 and 6.7 times, and the AUC_0–∞ ascending 4.4 and 10.8 times, respectively. When pretreated with the Oat inhibitor probenecid, the C_max increased 4.4 and 20 times, and the AUC_0–∞ increased 3.2 and 13.9 times, respectively. The results suggested that COG and COS may be the substrates of Oatp and Oat.

2. The accumulation of curcumin significantly increased in organic anion transporting polypeptide (OATP)- and organic anion transporter (OAT)-transfected human embryonic kidney (HEK) 293 systems, which suggested that curcumin was a substrate of OATP1B1, OATP1B3, OATP2B1, OAT1, and OAT3; and COG was a substrate of OATP1B1, OATP1B3, and OAT3.

3. Inhibition study using rosuvastatin as the substrate in OATP1B1- and OATP1B3-transfected cells indicated that curcumin was an OATP1B1 and 1B3 inhibitor, with IC₅₀ at 5.19?±?0.05 and 3.68?±?0.05?μM, respectively; the data for COG were 1.04?±?0.01 and 1.08?±?0.02?μM, respectively. COS was speculated to be an inhibitor of hepatic OATP1B1 as calculated using the ADMET Predictor.

4. COG and COS are substrates and inhibitors of OATP/Oatp. Co-administration of curcumin significantly increased rosuvastatin concentration in rat and dog plasma.