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1.
A library of tetrapeptides was evaluated for Hepatitis C Virus NS3 protease inhibitor activity in an in vitro assay system comprising the native bifunctional full-length NS3 (protease-helicase/NTPase) protein. Tetrapeptides with Ki values in the high nanomolar range were identified, for example Suc-Chg-Glu-2-Nal-Cys (Ki=0.27±0.03 μM) and Suc-Dif-Glu-Glu-Cys (Ki=0.40±0.10 μM). Furthermore, it was shown that the inhibitory potencies are not affected significantly by assay ionic strength. As suggested by molecular modelling, potential binding interactions of the tetrapeptide inhibitors with the helicase domain might explain the data and structure–activity relationships thus obtained. Hence, we postulate that the full-length NS3 assay is a relevant system for inhibitor identification, offering new opportunities for inhibitor design.  相似文献   

2.
Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids. In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase. The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin (Ki 53 μM) and ciprofibrate (Ki 1000 μM). Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform (Km 6 ± 2.5 μM, (Vmax 0.33 ± 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean Ki 1.7 μM, n = 5) and R-ibuprofen (mean Ki 10.8 μM, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors. The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (Vmax/Km 0.057 ± 0.011 nmol/mg/min ± M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes. Evidence of both a substantial difference between the Km and Ki for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct. Thus until the current ‘long-chain like’ xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates.  相似文献   

3.
A novel class of Cathepsin B inhibitors has been developed with a 1,2,4-thiadiazole heterocycle as the thiol trapping pharmacophore. Several compounds with different dipeptide recognition sequence (i.e., P1′–P2′=Leu-Pro-OH or P2–P1=Cbz-Phe-Ala) at the C5 position and with different substituents (i.e., OMe, Ph, or COOH) at the C3 position of the 1,2,4-thiadiazole ring have been synthesized and tested for their inhibitory activities. The substituted thiadiazoles 3a–h inhibit Cat B in a time dependent, irreversible manner. A mechanism based on active-site directed inactivation of the enzyme by disulfide bond formation between the active site cysteine thiol and the sulfur atom of the heterocycle is proposed. Compound 3a (Ki=2.6 μM, ki/Ki=5630 M−1 s−1) with a C3 methoxy moiety and a Leu-Pro-OH dipeptide recognition sequence, is found to be the most potent inhibitor in this series. The enhanced inhibitory potency of 3a is a consequence of its increased enzyme binding affinity (lower Ki) rather than its increased intrinsic reactivity (higher ki). In addition, 3a is inactive against Cathepsin S, is a poor inhibitor of Cathepsin H and is >100-fold more selective for Cat B over papain.  相似文献   

4.
3β-hydroxysteroid dehydrogenase 5-ene isomerase (3βHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3βHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (Km=0.17±0.03 and 1.02 μM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected Vmax while Km was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. Ki was calculated from replot of Dixon's slope for each substrate concentration. Ki from the intercept and the slope of this replot were similar (0.276±0.01 and 0.263±0.02 μM) and higher than the Km for DHEA. The Km and Ki values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the Vmax while Km values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.  相似文献   

5.
The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to ΔpH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (Ki = 12 μM) and ATPase activity of inverted inner membrane vesicles (Ki = 126 μM) and partially purified F1-ATPase (Ki = 177 μM). The smaller Ki for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 μM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. Δψ-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of Δψ in isolated mitochondria.  相似文献   

6.
Polyphosphates of different chain lengths (P3, P4, P15, P35), (1 μM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p4A and P4 (1 μM) was 40 and 60%, respectively, whereas 1 μM Ap4A was not inhibitory. P4 and P15 were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA. In (a), P4 and P15 behaved as competitive inhibitors, with Ki values of 0.5 μM and 0.2 μM, respectively. In addition, P4 (at 1 μM) and P15 (at 0.3 μM) changed the Hill coefficient (nH) from 1 (control) to about 1.3 and 1.6, respectively. In (b), the inhibition by P4 and P15 decreased V and modified only slightly the Km values of the enzyme towards tRNA.  相似文献   

7.
1H-Pyrazole-1-carboxamidines were prepared as potential inhibitors of the three isozymes of nitric oxide synthase. All of the compounds were found to be competitive inhibitors of all three isoforms. The most selective compound prepared was 1H-pyrazole-N-(3-aminomethylanilino)-1-carboxamidine (14), which is 100-fold selective for nNOS over eNOS with a Ki value of 2 μM.  相似文献   

8.
Steroid sulfatase (STS) catalyzes the hydrolysis of steroidal sulfates such as estrone sulfate (ES1) to the corresponding steroids and inorganic sulfate. STS is considered to be a potential target for the development of therapeutics for the treatment of steroid-dependent cancers. Two steroidal and two coumarin- and chromenone-based boronic acids were synthesized and examined as inhibitors of purified STS. The boronic acid analog of estrone sulfate bearing a boronic acid moiety at the 3-position in place of the sulfate group was a good competitive STS inhibitor with a Ki of 2.8 μM at pH 7.0 and 6.8 μM at pH 8.8. The inhibition was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. An estradiol derivative bearing a boronic acid group at the 3-position and a benzyl group at the 17-position was a potent reversible, non-competitive STS inhibitor with a Ki of 250 nM. However, its 3-OH analog, a known STS inhibitor, exhibited an almost identical affinity for STS and also bound in a non-competitive manner. It is suggested that these compounds prefer to bind in a hydrophobic tunnel close to the entrance to the active site. The coumarin and chromenone boronic acids were modest inhibitors of STS with IC50s of 86 and 171 μM, respectively. Surprisingly, replacing the boronic acid group of the chromenone derivative with an OH group yielded a good reversible, mixed type inhibitor with a Ki of 4.6 μM. Overall, these results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog.  相似文献   

9.
Cholinesterase inhibitory and spasmolytic potential of steroidal alkaloids   总被引:3,自引:0,他引:3  
A new steroidal alkaloid, isosarcodine (1) along with four known bases, sarcorine (2), sarcodine (3), sarcocine (4) and alkaloid-C (5) were isolated from the MeOH extract of Sarcococca saligna. The structures of these alkaloids were identified by spectral data interpretation. These compounds were subjected to acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition studies, and were found to be noncompetitive inhibitors of AChE (Ki = 21.8, 90.3, 32.2, 16.0 and 50.0 μM, respectively) and uncompetitive or noncompetitive inhibitors of BChE (Ki = 8.3, 7.5, 15.6, 5.0 and 12.0 μM, respectively).

The compounds (2–5) also showed dose-dependent spasmolytic activity in the rabbit jejunum intestinal preparations and also relaxed the high K+ (80 mM)-induced contraction, indicative of a calcium channel-blocking mechanism.

Structure–activity relationship suggested that the nitrogen substituents at C-3 and/or C-20 of steroidal skeleton and the hydrophobic properties of the pregnane skeleton are the key structural features contributed to the inhibitory potency of these steroidal alkaloids against AChE and BChE.  相似文献   


10.
N-Acyl-N-hydroxy-β-amino acid derivatives were prepared and tested as inhibitors for thermolysin to find that these inhibitors show the -stereospecificity in contrast to the corresponding hydroxamates prepared from -amino acid, which exhibit the -stereochemistry. N-Formyl-N-hydroxy-β- -Phe-NHMe is the most potent inhibitor having the Ki value of 1.66 μM.  相似文献   

11.
To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4–25-fold higher affinity for the Y2 receptor than for the Y1 receptor. The affinity of [Leu31,Pro34]NPY is 7–60-fold higher for the Y1 receptor when compared with the Y2 subtype. Species selectivity within the Y2 receptors is demonstrated by PYY(3–36), NPY(2–36), NPY(22–36), and NPY(26–36). It is shown that NPY(22–36) is species selective for the human Y2 subtype (Ki of 0.3 nM) compared with the rabbit and rat Y2 receptor (Ki of 2 and 10 nM, respectively). PYY(3–36) displays highest affinity for the human and rabbit Y2 subtype (Ki of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y2 receptor is shown by NPY(2–36) and PYY(3–36). In addition, PYY(3–36) and NPY(2–36) are not only subtype selective, but also species selective.  相似文献   

12.
The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3,5β-tetrahydrocortisol; 3,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6β-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [3H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The Km value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the Vmax value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole (Ki = 0.9 ± 0.4 μM; N = 4), followed by gestodene (Ki = 5.6 ± 0.6 μM) and cyclosporine (Ki = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition (Ki = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The Km value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM (n = 4) and the Vmax value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (Ki = 17.8 ± 3.3 μM) and gestodene (Ki = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.  相似文献   

13.
Heterodimeric compounds H-Dmt-Tic-NH-hexyl-NH-R (R = Dmt, Tic, and Phe) exhibited high affinity to δ- (Kiδ = 0.13–0.89 nM) and μ-opioid receptors (Kiμ = 0.38–2.81 nM) with extraordinary potent δ antagonism (pA2 = 10.2–10.4). These compounds represent the prototype for a new class of structural homologues lacking μ-opioid receptor-associated agonism (IC50 = 1.6–5.8 μM) based on the framework of bis-[H-Dmt-NH]-alkyl (Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki, Y.; Ambo, A.; Konishi, R.; Nagata, M.; Salvadori, S.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem. 2003, 46, 3201), which exhibited both high μ affinity and bioactivity.  相似文献   

14.
The hydrolysis of steroid sulphates, by steroid sulphatase, is an important source of oestrogenic steroids (oestrone, oestradiol and 5-androstene-3β,17β-diol) which are found in tumours. In the present study, we have examined the effect of dehydroepiandrosterone-3-O-methylthiophosphonate (DHA-3-MTP), pregnenolone-3-O-methylthiophosphonate (pregnenolone-3-MTP) and cholesterol-3-O-methylthiophosphonate (cholesterol-3-MTP) on the inhibition of oestrone sulphatase as well as DHA sulphatase activities in intact MCF-7 breast cancer cells and in placental microsomes. All three methylthiophosphonates significantly (P< 0.01) inhibited the hydrolysis of oestrone sulphate (E1 S) in intact MCF-7 cells (31–85% inhibition at 1 μM and 53–97% inhibition at 10 μM). Significant inhibition of DHA sulphatase was also achieved. At a concentration of 50 μM, all three compounds inhibited the hydrolysis of dehydroepiandrosterone sulphate (DHAS) by > 95%. Using human placental microsomes, the Km and Vmax of E1S were determined to be 8.1 μM and 43 nmol/h/mg protein. The corresponding Ki values for DHA-3-MTP, pregnenolone-3-MTP and cholesterol-3-MTP were found to be 4.5, 1.4 and 6.2 μM, respectively. Such inhibitors which are resistant to metabolism may have considerable potential as therapeutic agents and may have additional advantage over aromatase inhibitors in also reducing tumour concentrations of the oestrogenic steroid, 5-androstene-3β,17β-diol, by inhibiting the hydrolysis of DHAS.  相似文献   

15.
Steven C. Huber  Gerald E. Edwards   《BBA》1977,462(3):603-612
1. Mesophyll chloroplasts of the C4 plant Digitaria sanguinalis contain endogenous phosphoenolpyruvate which appears to distribute across the envelope according to the existing pH gradient. The phosphoenolpyruvate remaining in the stroma can be rapidly released by external inorganic phosphate or 3-phosphoglycerate while external pyruvate did not affect the distribution.

2. Phosphoenolpyruvate (PEP) was a competitive inhibitor (Ki(PEP) = 450 μM) of 32Pi uptake (Km(Pi) = 200 μM) by chloroplasts in the dark and also reduced the steady-state internal concentration of 32Pi, which is consistent with phosphate and phosphoenolpyruvate sharing a common carrier.

3. Phosphoenolpyruvate formation by chloroplasts in the light in the presence of pyruvate but in the absence of inorganic phosphate was slow and the concentration ratio of phosphoenolpyruvate (internal/external) was high. Addition of 0.1 mM phosphate induced a high rate of phosphoenolpyruvate formation and the concentration ratio (internal/external) decreased 15-fold. It is proposed that external phosphate is required both for phosphoenolpyruvate formation and efflux from the chloroplast.  相似文献   


16.
The present study was undertaken to characterize the binding activities of propiverine and its N-oxide metabolites (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide: P-4(N → O), 1-methyl-4-piperidyl benzilate N-oxide: DPr-P-4(N → O)) toward L-type calcium channel antagonist receptors in the rat bladder and brain. Propiverine and P-4(N → O) inhibited specific (+)-[3H]PN 200–110 binding in the rat bladder in a concentration-dependent manner. Compared with that for propiverine, the Ki value for P-4(N → O) in the bladder was significantly greater. Scatchard analysis has revealed that propiverine increased significantly Kd values for bladder (+)-[3H]PN 200–110 binding. DPr-P-4(N → O) had little inhibitory effects on the bladder (+)-[3H]PN 200–110 binding. Oxybutynin and N-desethyl-oxybutynin (DEOB) also inhibited specific (+)-[3H]PN 200–110 binding in the rat bladder. Propiverine, oxybutynin and their metabolites inhibited specific [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding in the rat bladder. The ratios of Ki values for (+)-[3H]PN 200–110 to [3H]NMS were markedly smaller for propiverine and P-4(N → O) than oxybutynin and DEOB. Propiverine and P-4(N → O) inhibited specific binding of (+)-[3H]PN 200–110, [3H]diltiazem and [3H]verapamil in the rat cerebral cortex in a concentration-dependent manner. The Ki values of propiverine and P-4(N → O) for [3H]diltiazem were significantly smaller than those for (+)-[3H]PN 200–110 and [3H]verapamil. Further, their Ki values for [3H]verapamil were significantly smaller than those for (+)-[3H]PN 200–110. The Ki values of propiverine for each radioligand in the cerebral cortex were significantly (P < 0.05) smaller than those of P-4(N → O). In conclusion, the present study has shown that propiverine and P-4(N → O) exert a significant binding activity of L-type calcium channel antagonist receptors in the bladder and these effects may be pharmacologically relevant in the treatment of overactive bladder after oral administration of propiverine.  相似文献   

17.
A series of methionine analogues have been synthesized as inhibitors of methionyl-tRNA synthetase and evaluated for their inhibitory activities of E. coli methionyl-tRNA synthetase and bacterial growth. Among them, -methionine hydroxamate 20 has proved to be the best inhibitor of the enzyme with Ki = 19 μM and showed a growth inhibition against E.coli JM 109, P. vulganis 6059 and C. freundii 8090.  相似文献   

18.
Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. S6-(4-nitrobenzyl)mercaptopurine riboside (NBMPR) is a prototype inhibitor of the human equilibrative nucleoside transporter (hENT1), and is a high affinity ligand with a Kd of 0.1–1.0 nM. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel C2-purine position substituted analogs of NBMPR at the hENT1. The aim of this research was to understand the substituent requirements at the C2-purine position of NBMPR. Structure–activity relationships (SAR) indicate that increasing the steric bulk at the C2-purine position of NBMPR led to a decrease in binding affinity of these ligands at the hENT1. New high affinity inhibitors were identified, with the best compound, 2-fluoro-4-nitrobenzyl mercaptopurine riboside (7), exhibiting a Ki of 2.1 nM. This information, when coupled with the information obtained from other structure–activity relationship studies should prove useful in efforts aimed at modeling the NMBPR and analogs pharmacophore of hENT1 inhibitors.  相似文献   

19.
The photoreactive nucleotide 3'-O-(4-benzoyl)benzoyl ADP (BzADP) is not a substrate for photophosphorylation but is a strong competitive inhibitor (Ki 2-25μM) with respect to ADP and ATP in photophosphorylation or ATP hydrolysis and Pi-ATP exchange reactions, respectively. The analog binds tightly to the membrane-bound CF1, competes with the right binding of ADP, and prevents the inactivation of the enzyme by tight binding of ADP. Upon irradiation with long wavelength ultraviolet light, the tightly bound BzADP becomes covalently attached to both the - and β-subunits of the enzyme.  相似文献   

20.
N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R′ series of compounds produced no significant affect on the high δ-opioid receptor affinity (Ki=0.035–0.454 nM), but dramatically decreased that for the μ-opioid receptor. The effect of N-methylation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the third aromatic group (R′): phenyl (Ph) (5′–8′) elicited a greater reduction in μ-affinity (40–70-fold) compared to analogues containing 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i) a loss of δ-agonism coupled with the appearance of potent δ antagonism (4′–7′) (pA2=8.14–9.47), while 1 exhibited only a 160-fold decreased δ agonism (1′) and the δ antagonism of 8 enhanced >10-fold (pA2=10.62, 8′); and (ii) a consistent loss of μ-affinity resulted in enhanced δ-opioid receptor selectivity. With the exception of compound 1′, the change in the hydrophobic environment at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced potent δ-selective antagonists.  相似文献   

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