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Kinetics Studies on Symplocomoside: A Urease Inhibitor.

Byline: MUHAMMAD ATHAR ABBASI, MUHAMMAD ARIF LODHI, AZIZ-UR-REHMAN, VIQAR UDDIN AHMAD AND MUHAMMAD IQBAL CHOUDHARY

Summary: The mechanism of inhibition of jack bean and Bacillus pasteurii ureases was investigated by symplocomoside (1) which is a phenolic glycoside of salirepin series and has been isolated from Symplocos racemosa Roxb. Lineweaver-Burk, Dixon plots and their secondary replots showed that 1 was a non-competitive inhibitor of these enzymes. Ki values were found to be 77.60 +- 1.22 uM and 63.47 +- 0.92 uM against jack bean and Bacillus pasteurii ureases respectively.

Introduction

Symplocos racemosa Roxb (Symplocaceae) is known as "Lodhra" and is used in the Indian system of medicine as single drug or in multicomponent preparations (viz., "Lodhrasava"). Medicinally the bark is used as an acrid, digestible, astringent for the bowels and is useful in eye diseases, spongy gums and for bleeding. The bark also shows depressant action on blood pressure and intestinal movements and is widely used as an Ayurvedic remedy mainly for gynecological disorders and for ulcers of vagina. Unani medicine uses it as emmenagogue and aphrodisiac. It is a potent remedy for inflammation and cleaning uterus and is also used to treat leucorrhea and menorrhagia [1]. The study on the bark of Symplocos racemosa Roxb. revealed that its aqueous extract on oral administration significantly stimulated serum follicle- stimulating hormone (FSH) level (P less than 0.016) along with the rise in serum luteinizing hormone (LH) level (P less than 0.001).

Moreover, histopathological studies revealed enhanced folliculogenisis, presence of mature follicles and detached oocytes, which were the result of increased FSH and LH levels. All these results were in concordance with the traditional use of this plant for female disorders [1]. Urease (urea amidohydrolase, EC: 3.5.1.5) occurs throughout the animal and plant kingdom. Many microorganisms use this enzyme to provide a source of nitrogen for growth and the enzyme plays an important role in plant nitrogen metabolism during the germination process [2, 3]. The presence of urease activity in soils is exploited in the widespread agricultural practice of urea-based fertilizer application for enhancing crop yields. Unfortunately, excessive levels of soil urease can degrade fertilizer urea too rapidly and result in phytopathic effects and loss of volatilized ammonia [4].

Of medical and veterinary interest, urease is a virulence factor in certain human and animal pathogens; it participates in the development of kidney stones, pyelonephritis, peptic ulcers and other disease states [5]. The obvious remedy for treating bacterial infection with antimicrobials, however, has often proven futile [6] and only a few combination regiments have reached clinical practice. Thus the need for alternative or novel treatment is evident. Consequently, the discovery of potent and safe urease inhibitors has been a very important area of pharmaceutical research due to the involvement of ureases in different pathological conditions. We have previously reported a number of novel synthetic and natural inhibitors of urease, their inhibition kinetics and structure-activity relationship studies [7-10].

In continuation of this work to discover new and potent inhibitors of medicinally important enzymes through high-throughput screening assays, we identified symplocomoside (1) as an effective inhibitor of jack bean and Bacillus pasteurii ureases. The objective of the current investigation is to explore the possible binding interactions of 1 in the target protein in search for an effective inhibitor of urease.

Results and Discussion

Symplocomoside (1) which is a phenolic glycoside of salirepin series was isolated as a white powder from the ethyl acetate soluble fraction of Symplocos racemosa Roxb. and its structure (Fig. 1) was deduced through extensive spectral studies [11].

Urease is an enzyme that is present in many plants and in soil that catalyzes the hydrolysis of urea to ammonium and carbamate ions, which decompose to carbon dioxide and ammonia. The active site contains two nickel (II) atoms which, as shown by X- ray analysis, are linked by a carbamate bridge; furthermore, two imidazole nitrogen atoms are bound to each nickel atom and a carboxylate group and a water molecule fill the remaining coordination site of the metal ion [5]. The coordination geometry of the first nickel atom is pseudo tetrahedral, while that of second is roughly trigonal bipyramidal. In order to discriminate among the inhibition capacities of various compounds, it is important to understand the coordination mechanism between the active site of the enzyme and the inhibitor.

Symplocomoside (1) inhibited urease enzymes in a concentration-dependent manner with Ki values of 77.60 +- 1.22 uM and 63.47 +- 0.92 uM against jack bean and Bacillus pasteurii ureases, respectively (Table-1). Ki values were calculated in three different ways; first, the slopes of each line in the Lineweaver-Burk plot were plotted against different concentrations of 1; second, the 1/Vmaxapp was calculated by plotting different fixed concentrations of urea versus [?]V in the presence of different fixed concentrations of 1 in the respective assays of urease. Then Ki was calculated by plotting different concentrations of inhibitor versus 1/Vmaxapp. Ki was the intercept on the x-axis. In the third method, Ki was directly measured from Dixon plot as an intercept on x-axis (Fig. 2). Determination of the inhibition type is important in understanding the mechanism of inhibition and the sites of inhibitor binding.

Lineweaver-Burk, Dixon plots and their replots indicated that 1 is a non-competitive inhibitor of jack bean and B. pasteurii ureases, as in its presence, there was a decrease in Vmax without affecting the affinity (Km values) of the urease towards the substrate (urea). In other words, 1 and urea bind randomly and independently at the different sites of urease indicating that inhibition depends only on the concentration of 1 and dissociation constant (Ki). These mechanistic studies of 1 are expected to provide rational information for the design of a new potential inhibitor of jack bean and B. pasteurii ureases.

A. Lineweaver- Burk plot of reciprocal of initial velocities versus reciprocal of four fixed J.B urease concentrations in absence and presence of 50.0 uM, 75.0 uM, 100 uM of 1. B is the Dixon plot of reciprocal of the initial velocities versus

various concentrations of 1 at fixed urease concentrations, 24 uM, 18.0 uM, 12.5 uM and 6.2 uM. C. is Dixon Secondry plot slope vs three different

Table-1: In vitro inhibition of ureases by symplocomo-side (1). (a) Standard mean error of 3-5 assays, (b) Ki is the mean of three values calculated by using the Dixon plot and Lineweaver-Burk secondary plots, (c) J.B is jack bean urease and B.P is Bacillus pasteurii urease

Substance###Enzymec###Type of Inhibition###Ki +- SEM Symplocomoside (1)###

###J.B urease###Non-competitive###77.60 +- 1.22

###B.P urease###Non-competitive###63.47 +- 0.92

Thiourea (Standard)###J.B urease###Competitive###18.15 +- 0.77

###B.P urease###Competitive###15.38 +- 0.39

Experimental

Our research group has already published the isolation and structure elucidation of symplocomoside (1) from Symplocos racemosa Roxb [11].

Urease Assay and Inhibition

Reaction mixtures comprising 25 uL of enzyme (jack bean urease and Bacillus pasteurii urease separately) solution and 55 uL of buffers containing urea (2-24 mM) were incubated with 5 uL of 1 at 30oC for 15 min in DMSO in 96-well plates. The increased absorbance at 560 nm was measured after 10 min., using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 uL. The results (change in absorbance per min) were processed by using SoftMax Pro software (Molecular Device, USA). All the assays were performed at pH 6.8 (3 mM sodium phosphate buffer) and 7 ug of phenol red per ml as indicator. Percentage inhibitions were calculated from 100-(ODtestwell/ODcontrol) x 100. Thiourea was used as the standard inhibitor of urease.

Determination of Kinetic Parameters

The concentration of 1 that inhibited the hydrolysis of substrates (jack bean and Bacillus pasteurii ureases separately) by 50% (IC50) was determined by monitoring the inhibition effect of various concentrations of 1 in the assay. The IC50 (inhibitor conc. that inhibits 50% activity of both enzymes) values were then calculated using the EZ- Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA). The interaction of 1 with jack bean and Bacillus pasteurii ureases can be described by the following scheme:

where ES is the jack bean and B. pasteurii urease- urea complex and P is the product. KI and bKI are the inhibition constants reflecting the interactions of 1 with the free J.B. and B.P. enzymes and enzymes- urea complex [12-14].

Statistical Analysis

Graphs were plotted using GraFit program [15]. Values of the correlation coefficients, slopes, intercepts and their standard errors were obtained by the linear regression analysis using the same program. The correlation for all the lines of all graphs was found to be greater than 0.99. Each point in the constructed graphs represents the mean of three experiments.

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1MUHAMMAD ATHAR ABBASI, 2MUHAMMAD ARIF LODHI, 1AZIZ-UR-REHMAN, 3VIQAR UDDIN AHMAD AND 3MUHAMMAD IQBAL CHOUDHARY 1Department of Chemistry, GC University, Lahore-54000, Pakistan. 2Department of Chemistry, Abdul Wali Khan University of Mardan, Mardan, Pakistan. 3HEJ Research Institute of Chemistry, International Centre for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. 1atrabbasi@yahoo.com*, 3vuahmad@hotmail.com*
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Author:Abbasi, Muhammad Athar; Lodhi, Muhammad Arif; Aziz-Ur-Rehman; Ahmad, Viqar Uddin; Choudhary, Muhamma
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:9PAKI
Date:Feb 29, 2012
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