Flavonoid Inhibitors of Trypsin and Leucine Aminopeptidase: A Proposed Mathematical Model for IC<sub>50</sub> Estimation

Parellada, Josefina; Guinea, M. Torres

doi:10.1021/np50120a001

Cited by 31 publications

(21 citation statements)

References 15 publications

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“…In the present study, the¯avonoids quercetin and kaempferol caused a dose-dependent linear inhibition of NPA binding, suggesting either a weaker or, more likely, a non-competitive interaction with the proposed NPAbinding site on the aminopeptidase. The inhibitory eects of dihydrochalcones and¯avonoids on a number of animal APs is well documented (Parellada 1995). We previously showed that Tyr-, Pro-, and Trp-AP activities are strongly inhibited by genestein, quercetin, and phloretin ±¯avonoids known to displace NPA from its binding site (Murphy and Taiz 1999b).…”

Section: Discussionmentioning

confidence: 98%

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

Murphy

Peer

Taiz

2000

Planta

316

293

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The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsis thaliana (L.) Heynh. seedlings. Moreover, amino acyl-beta-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [3H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [14C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux.

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Section: Discussionmentioning

confidence: 98%

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

Murphy

Peer

Taiz

2000

Planta

316

293

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“…Cheburator supports three approaches – the simple two-points method previously described by Reed and Muench [7], a linear regression, and a nonlinear regression. The default method is nonlinear regression, which is currently considered to be “state-of-the-art” in the field of experimental drug screening [5], [8], [9].…”

Section: Methodsmentioning

confidence: 99%

Cheburator Software for Automatically Calculating Drug Inhibitory Concentrations from In Vitro Screening Assays

Nevozhay

2014

PLoS ONE

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In the process of new cancer drug development, as the first step of their assessment, their activities are usually studied in vitro against a panel of cancer cell lines. The results of these in vitro drug screening assays are commonly expressed as inhibitory concentration 50% (IC50): the concentration of the tested agent that inhibits the proliferation of the cancer cell population to 50% of the theoretically possible effect (absolute IC50) or maximum effect practically achieved by the drug (relative IC50). The currently available software for calculating IC50 values requires manual data entry, is time consuming, and is prone to calculation errors. Thus, we have developed open source, free, easy-to-use software for performing standardized data evaluations and automatically calculating the IC50. This software eliminates the laborious and error-prone manual entry of data, substantially reduces the amount of time spent for data analysis. It has been extensively used in our department as the main tool for in vitro data processing during the past several years and can be useful for other research groups working in the area of anticancer drug discovery, either alone or combined with other software packages. The current version of our program, Cheburator, together with sample data, source code, and documentation, is freely available at the following URL: http://www.cheburator.nevozhay.com (it is free for academic use, but a license is required for commercial use).

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“…Flavonoids especially kaempferol, were found to have inhibitory effects on trypsin as a result of their hydroxyl groups [27]. In serum albumin strongly by hydrophobic interactions and this resulted in changes in secondary structure of protein [22].…”

Section: Trypsin Stability Studiesmentioning

confidence: 99%

Kinetic and structural characterization of interaction between trypsin and Equisetum arvense extract

Uslu¹,

Bayraktar²,

Ceylan³

2014

Turk J Bioch

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Kinetic and structural characterization of interaction between trypsin and Equisetum arvense extract [Trypsin ve Equisetum arvense özütü arasındaki etkileşimin kinetik ve yapısal karakterizasyonu] ABSTRACT Objective: In this study the inhibitory effect of E. arvense extract on trypsin activity and the effect of trypsin on E. arvense extract were studied. In addition the nature of the interaction between the extract and trypsin was investigated. Methods: The inhibitory effect ethanol extract of E. arvense on trypsin activity was determined using trypsin enzyme assay. The structural effects of the extract-trypsin interaction for the extract were analyzed by FTIR. Finally, the HPLC analyses were carried out to analyze the individual components of the extract and the supernatant and soluble precipitate phases. Results: E. arvense extract was found to decrease total percent activity of trypsin to 5% in 24 hour at 24 °C. FTIR analyses indicated that the interaction between trypsin and E. arvense extract caused changes in the structure and hydrogen bonding behavior and composition of the extract proteins. These interactions also caused the extract lipids to accumulate in the insoluble precipitate phase. Most of the phenolics remained in the supernatant phase enhancing the inactivation of trypsin. However, the precipitated compounds were shown to be of apolar in nature as shown in the HPLC chromatograms. Conclusion:The methods that were used showed that the high phenolic content of E. arvense was the main reason for the inhibition of trypsin enzyme activity by denaturing the enzyme. of 1:20. The dry leafs particles were shaked in the solution for overnight at 300 rpm at 24 °C. After the centrifugation at 2000xg for 5 minutes the supernatant was collected into 50 ml falcon tubes. The alcohol in the liquid phase was evaporated with a rotary evaporator. The aqueous extract was frozen for 24 hours and then freeze dried in a lyophilizer (Telstar-Cryodos). Preparation of inhibition solutionE. arvense extract was dissolved in distilled water to a final concentration of 10 mg/ml. By serial dilutions of stock solution 2 mg/ml, 0.2 mg/ml and 0.02 mg/ml concentrations were prepared. Preparation of trypsin solutionBovine pancreatic trypsin was purchased from Fluka Analytical (cat.93610) and used without further purification. Lyophilized powder of bovine pancreatic trypsin (Fluka Analytical, USA) was dissolved in 1 mM HCl to a final concentration of 0.1 mg/ml. Determination of enzyme kineticsTrypsin catalyzes the hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE) to N-benzoyl-L-arginine (BA) and ethanol. The slope of the absorbance versus time graph was the production rate of BA at pH 7.6.The reaction was held in a total volume of 3.2 ml quartz cuvette at 25 °C. The unit of trypsin activity was previously determined as 1 µmol BA produced per minute at 253 nm, at pH 7.6 and 25 °C. The reaction was carried out in 100 mM Tris buffer solution. To determine absorption of blank solution, 3050 μl tris buffer and 150 μl of BAEE solution ...

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Flavonoid Inhibitors of Trypsin and Leucine Aminopeptidase: A Proposed Mathematical Model for IC₅₀ Estimation

Cited by 31 publications

References 15 publications

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

Cheburator Software for Automatically Calculating Drug Inhibitory Concentrations from In Vitro Screening Assays

Kinetic and structural characterization of interaction between trypsin and Equisetum arvense extract

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Flavonoid Inhibitors of Trypsin and Leucine Aminopeptidase: A Proposed Mathematical Model for IC50 Estimation

Cited by 31 publications

References 15 publications

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

Cheburator Software for Automatically Calculating Drug Inhibitory Concentrations from In Vitro Screening Assays

Kinetic and structural characterization of interaction between trypsin and Equisetum arvense extract

Contact Info

Product

Resources

About

Flavonoid Inhibitors of Trypsin and Leucine Aminopeptidase: A Proposed Mathematical Model for IC₅₀ Estimation