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Biochemical and Biophysical Research Communications 356 (2007) 457–463

Germa-c-lactones as novel inhibitors of bacterial urease activity

Zareen Amtul a,*,1, Cristian Follmer b, Sumera Mahboob c, Atta-Ur-Rahman a,Muhammad Mazhar c, Khalid M. Khan a, Rafat A. Siddiqui a,d, Sajjad Muhammad e,

Syed A. Kazmi a,f, Mohammad Iqbal Choudhary a,*

a International Center for Chemical Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistanb Departamento de Fısico-Quımica, Instituto de Quımica, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-909, Brazil

c Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistand Methodist Research Institute, 1701 N. Senate Ave, Indianapolis, IN 46202, USA

e Department of Neurologie, Im Neuenheimer Feld 400, Heidelberg University, Heidelberg 69120, Germanyf Department of Chemistry, University of Karachi, Karachi 75270, Pakistan

Received 27 February 2007Available online 8 March 2007

Abstract

Organogermanium compounds have been used as pharmacological agents. However, very few reports are available on the synthesisand antibacterial activities of lactones containing organogermaniums. The purpose of the present investigation was to determine theeffects of different lactone-substituted organogermaniums on bacterial growth and their urease activity. We report synthesis of 12germa-c-lactones (GeL) and their antimicrobial activities against several bacterial pathogens. Antibacterial action of all GeL washighly selective against Gram-negative bacilli, particularly Proteus mirabilis, an important pathogen infecting the urinary tract. Fur-thermore, our data indicate that 8-quinoline derivatives were more potent against P. mirabilis than 2-methyl-8-quinoline. For example,the b-(o-methylphenyl)-c,c-bis(8-quinolinoxy)germa-c-lactone and b-(o-methoxyphenyl)-c,c-bis(8-quinolinoxy)germa-c-lactone weremaximally active with MIC90 of 61 and 94 lM, respectively. In vitro studies demonstrated a linear correlation between antibacterialactivity and inhibition of P. mirabilis urease enzyme. Further kinetic analyses revealed that inhibition occurred in a noncompetitiveand concentration-dependent manner with the minimum IC50 of 31 lM for b-(o-methoxyphenyl)-c,c-bis(8-quinolinoxy)germa-c-lac-tone. In conclusion, these findings suggest that GeL have potential to be developed as antimicrobial agents against P. mirabilis

infection.� 2007 Elsevier Inc. All rights reserved.

Keywords: Germa-c-lactones; Proteus mirabilis; Urease; Noncompetitive inhibitor; Enzyme kinetics; Enzyme inhibition

Inorganic germanium salts and novel organogermaniumcompounds including carboxyethyl germanium sesquioxide(Ge-132) and lactate–citrate–germanate (Ge lactate citrate)are used as ‘‘nutritional supplements’’ because of theirimmunomodulatory effects [1]. Other organogermaniuncompounds such as germatranes, spirogermanium, 2-carb-

0006-291X/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2007.02.158

* Corresponding authors. Fax: +92 21 4819018 19.E-mail addresses: zareen_amtul@fulbrightweb.org, zamtul@uwo.ca

(Z. Amtul), hej@cyber.net.pk (M.I. Choudhary).1 Present address: Department of Biochemistry, University of Western

Ontario, Canada.

oxyethyl germanium sesquioxide, carboxyethyl germaniumsesquisulfide and germa-c-lactones have been reported fora variety of activities including their possible use in sup-pression of the growth of certain tumors, proliferation ofthe normal marrow cells in the tumor bearing animals, inpain relief, immunomodulation, induction of interferon,regulation of plant growth, hepatic cirrhosis, cardiovascu-lar function, motor activity and stimulation of red bloodcells production [2–8]. The 8-quinolinol (one of the lactonesubstituted compounds) and its derivatives are widely usedas, antiamoebic and antiseptic agents [9]. Furthermoremetal chelation has been implicated in the biological

458 Z. Amtul et al. / Biochemical and Biophysical Research Communications 356 (2007) 457–463

activity of derivatives of oxine (e.g., some medicines likeVioform (Ciba), 5-chloro-7-iodo-8-hydroxyquinoline usedin the treatment of gastrointestinal infection and (Ox)2Cupossesses fungicidal activity) [10].

The purpose of this study was to investigate the effects ofdifferent lactones substituents on the germyl moiety oforganogermaniums in order to improve their bactericidaleffects. In the present study, we evaluated the antimicrobialactivities of lactones containing organogermaniun com-pounds. Based on the scaffold (Fig. 1, inset), we designedand synthesized from a synthetic combinatorial library,twelve different germa-c-lactones (GeL; denoted as 1–12)with hexacoordinated Ge centre, which is bonded to twoN atoms, three O atoms and one C atom, yielding a slightlydistorted octahedral geometry. These GeL showed selectiveantibacterial activities against a number of urease produc-ing bacteria notably Proteus mirabilis. Furthermore, a lin-ear correlation between urease inhibitory activity andantibacterial activity of GeL was observed. This impliesthat germa-c-lactones may confer antibacterial activityagainst P. mirabilis by inhibiting its urease enzyme. Thekinetic analyses revealed that inhibition occurred in a non-competitive and concentration-dependent manner. Givenboth the importance of urease for infection by P. mirabilis

and the enhanced inhibition of the enzyme by GeL, we con-clude that GeL may exert their antibacterial action throughthe inhibition of urease.

RCOOH

GeCl3

NO

NO

GeO

R

NO

NO

GeO

O

R

NOH

O

NH3, M

Fig. 1. Synthetic scheme germa-c-lactones. GeL were synthesized by the reactmethyl-8-quinolinol in dry MeOH under reflux in the presence of concentrate

Materials and methods

Chemical synthesis. Germa-c-lactones (Fig. 1) were synthesized by thereaction of substituted trichlorogermyl propionic acids (11.27 mmol, 1equivalent), of general formulae GeCl3CH(R)CH2CO2H where R = CH3,C6H5, o-CH3C6H4, p-CH3C6H4, o-OCH3C6H4, p-OCH3C6H4, with8-quinolinol or 2-methyl-8-quinolinol (33.81 mmol, 3 equivalents) in dryMeOH (70 ml) under reflux in the presence of concentrated NH3 (1.8 ml)as described [11], see the Supplemental file for spectroscopic character-ization. The resultant solid product was filtered, washed with MeOH forseveral times and dried under vacuum. The respective substituted tri-chlorogermyl propionic acids were prepared as described [12–16].Elemental microanalysis (C, H, N percentages), Fourier transformedInfrared (FTIR), 1H and 13C NMR and mass spectrometry were used todetermine the structures of compounds. The structure of compound 3 asrepresentative of the series has previously been characterized by X-raycrystallography [11].

Bacterial strains. Seven bacterial species used in this study are listed inTable 1. All cultures were clinical isolates obtained from Liaquat NationalHospital, Karachi, Pakistan and grown at 37 �C using standard proce-dure. P. mirabilis strain was isolated from urinary-tract infection, pri-marily from urolithiasis. Isolates were identified using standard methods[17] and were maintained frozen at �80 �C until further use for experi-ments. The standard P. mirabilis strain (ATCC 29855) was used forexperimental and quality control purposes.

In vitro susceptibility testing. In vitro susceptibility testing was deter-mined by adding 5 ll of test compounds (dissolved in DMSO) to 95 ll ofthe microbial suspensions of 107–8 colony forming unit (CFU)/mL in anutrient broth containing yeast extract (2%), NiCl2 (1 mM) and urea (2%)in 96-well plates. The bacterial cultures were incubated at 37 �C for 24 h.The progression in the turbidity of the broth was used as a measure ofbacterial growth and measured at 600 nm in a 96-well Microplate Reader

NH4ClNH4(Ox)+

(HOx)

NOH

etOH

N

O

O

Ge

O

R

2

ion of substituted trichlorogermyl propionic acids with 8-quinolinol or 2-d NH3. The inset shows the scaffold used to design and synthesize GeL.

Tab

le1

Th

eM

IC(l

M)

valu

eso

fge

rma-

c-la

cto

nes

agai

nst

diff

eren

tb

acte

rial

spec

ies

Str

ain

s(r

od

s)1

23

45

67

89

10

11

12

Baci

llus

subti

lis

721

±62

1156

±10

766

0.1

±51

1226

±11

242

7±

3277

7±

5511

86±

5713

64±

9310

60±

126

1074

±96

1095

±97

1393

±99

Baci

llus

past

euri

i74

1.6

±64

1079

±99

678.

96±

5212

61±

116

439.

2±

2879

9.2

±33

1425

.6±

5910

03±

117

1097

±90

1310

.4±

9911

22±

121

1132

.8±

102

Esc

her

ich

iaco

li10

21±

9216

50±

157

942

±79

1751

±16

561

0±

4511

10±

5119

80±

9119

50±

173

1801

±18

318

20±

168

1851

±17

019

91±

173

Kle

bsi

ella

pn

eum

on

iae

360.

5±

2656

7.5

±48

330.

05±

1861

2.5

±51

213.

4±

838

8.5

±11

694

±18

682.

5±

4462

0±

5862

7±

5264

7.5

±52

686.

5±

54.5

Pse

ud

om

on

as

aer

ug

inosa

772.

5±

6712

37.5

±11

570

7.25

±55

1313

±12

145

8.5

±31

832.

5±

3510

86±

6311

62.5

±83

1150

±10

113

65±

100

1387

.5±

121

1292

.5±

123

Pro

teu

svu

lag

ris

357.

5±

2551

2.5

±43

335.

75±

1843

7.5

±33

252.

5±

1037

7.5

±15

495

±17

487.

5±

2445

0±

3845

5±

3546

2.5

±35

497.

5±

36P

rote

us

mir

abil

is10

3±

0.3

165

±8.

094

.3±

917

5±

7.5

61±

4.1

111

±6.

119

8±

4.1

195

±9.

818

0±

9.5

182

±8

185

±8.

219

9±

8.5

MIC

,m

inim

um

inh

ibit

ory

con

cen

trat

ion

.

Z. Amtul et al. / Biochemical and Biophysical Research Communications 356 (2007) 457–463 459

(Molecular Devices, USA). Minimum inhibitory concentrations (MICs):the lowest concentrations of the compound giving >90% inhibition ofbacterial growth were determined in the presence of 10–500 lM GeL after24 h of incubation. Ceftriaxone (Sigma), a standard antibiotic, was used aspositive control to evaluate bacterial growth inhibition.

Urease inhibition assay. Proteus mirabilis urease (PMU) was purified asdescribed [18]. The specific activity of the enzyme was 100,000 U/g pro-tein. The ureases from Bacillus pasteurii and jack beans were purchasedfrom Sigma and used as controls. In order to evaluate the urease inhibi-tory properties of GeL, 25 ll of purified enzymes (0.5 mg protein/mlsolutions) were incubated with 5 ll of GeL (10–500 lM, in DMSO) at37 �C, pH 7.5 (0.01 M K2HPO4, 1 mM EDTA and 0.01 M LiCl2) for30 min in 96-well plates. All the assays were performed at pH 7.5 exceptfor the determination of the Ki and IC50 values that were performed at pH5.6 and 8.2. The urease enzymes have a pH range of 5.0–10.6 with optimalactivity at 7.5 [19]. Urease activity was determined by measuring NH3

production using the indophenol method [20]. One unit of urease wasdefined as the amount of enzyme that releases one lmol of NH3 perminute, at 37 �C, pH 7.5. The concentration that inhibited 50% of ureaseactivity (IC50) was determined using the EZ-Fit� 5.0 program (PerrellaScientific Inc., USA). Hydroxyurea (Sigma) was used as standard ureaseinhibitor.

DNA damaging assay. In order to evaluate the potential of GeL toinduce DNA damage, the compounds were incubated with Saccharomyces

cerevisiae strains (gifted by Leo F. Faucette of Smith Kline & FrenchResearch & Development Labs, PA, USA) in media enriched with 1%yeast extract, 2% peptone, and 2% dextrose (YPD media) with or without2% agar as described [21]. The progression in the turbidity of the brothwas used as a measure of yeast growth and measured at 600 nm in a 96-well Microplate Reader (Molecular Devices, USA).

Estimation of kinetic parameters. The Ki values (the dissociation/inhi-bition constant of the urease-GeL complex into free PMU and GeL) werecalculated using three different methods. Initially, the slope of eachLineweaver–Burk (LWB) plot was plotted against concentrations of dif-ferent GeL. Second, 1/Vmax(app) was calculated at each concentration ofdifferent GeL and then plotted against GeL concentration; the Ki wascalculated from the abscissa. Finally the Ki were determined directly fromthe intersections of the line for each substrate concentration on the x-axis.Similarly, Vmax(app) was determined by the intersection of the line for eachsubstrate concentration on the y-axis. The results (change in absorbanceper minute) were processed using SoftMax Pro software.

Estimation of protein concentration. Protein content of the enzymepreparation was estimated as described by Lowry [22], using bovine serumalbumin (Sigma) as standard.

Statistical analysis. Graphs were plotted by using GraFit program(Version 4.09, Erithacus Software Ltd., Staines, UK). Values includingMIC, IC50 Km, Ki, Vmax, and their standard errors are presentedas means ± SEM and obtained by the linear regression analysis. Allstatistical comparisons were by two-tailed t-test. Significance was set atp < 0.05.

Results and discussion

Germanium is found in all living matters in micro-tracequantities. Its therapeutic attributes include immuno-enhancement, oxygen enrichment, and free radical scaveng-ing, analgesia and heavy metal detoxification. Use of ger-manium in clinical trials has demonstrated its efficacy intreating a wide range of serious afflictions, including can-cer, arthritis and senile osteoporosis [23].

The purpose of this study was to investigate the effects ofdifferent lactones substituents on the germyl moiety oforganogermaniums to extend their antibacterial activityand potency. In this study we synthesized and evaluatedtwelve germa-c-lactones (GeL) for their antimicrobial

30 40 50 60 70 80 90 100 110 120

60

80

100

120

140

160

180

200

2-methyl-8-quinolines8-quinolines

20 40 60 80 100 120 14040

60

80

100

120

140

160

180

200

220

Antib

acte

rial a

ctiv

ity a

gain

stP.

mira

bilis

, MIC

(μM

)

Urease inhibition, IC50 (μM)

R2=0,956

Fig. 2. Correlation of antibacterial activity of GeL against P. mirabilis

(expressed as MIC, lM) with PMU (expressed as IC50, lM). The insetshows the activities of 8-quinolines and 2-methyl-8-quinolinesderivatives.

460 Z. Amtul et al. / Biochemical and Biophysical Research Communications 356 (2007) 457–463

potential. Our data indicates that GeL has antibacterialactivity against a wide range of Gram-negative bacilli (suchas Klebsiella pneumoniae, P. mirabilis, and P. vulgaris)(Table 1). In particular, the effect of GeL was very signifi-cant on P. mirabilis, whereas a weak effect was found onother bacilli. This selectivity suggests that P. mirabilis ispotentially vulnerable to GeL compounds.

We next analyzed the minimal inhibitory concentrations(MIC) to inhibit the 90% growth of the clinical isolate of P.

mirabilis. We observed that all GeL compounds inhibitedP. mirabilis growth significantly compared to ceftriaxone,where the GeL 5, 8 and 9 (8-quinoline derivatives) werethe most potent compounds (Table 2).

Proteus mirabilis causes urinary tract infection. The vir-ulence of P. mirabilis is mediated through its urease enzymeactivity which also causes urolithiasis [24]. Studies haveshown that a urease-negative mutant of P. mirabilis wasunable to initiate stone formation and colonization in thekidney at a significantly lower rate [25]. Given boththe importance of urease for infection by P. mirabilis andthe enhanced inhibition of the GeL compounds againstP. mirabilis, we hypothesized that GeL may exert theirantibacterial action through the inhibition of urease.Therefore, in order to get an insight into the mechanismof antibacterial activity of GeL, we tested the effect ofGeL on P. mirabilis urease (PMU). Our data suggest thatGeL compounds have significant inhibitory effect onPMU activity compared to hydroxyurea, a widely usedurease inhibitor (Table 2).

GeL compounds inhibited PMU in a concentration-dependent manner with Ki values in the range of 15.6 (com-pound 6) to 92.2 lM (compound 7) (Table 2). This resultsuggests that the inhibitory action of GeL compounds onthe growth of P. mirabilis might be mediated through inhi-bition of urease enzyme. The data in Fig. 2 exhibits a linearcorrelation (R2 = 0.956) between the antimicrobial andPMU inhibitory activity of GeL. These results are similarand consistent with the previous finding that showed a

Table 2Effects of germa-c-lactones on PMU activity, Proteus mirabilis and Saccharom

GeL Ligand R PMU inhibition

IC50 (lM) Ki (l

Standard — — 150 ± 5 hydroxyurea —

1 L1 CH3 65 ± 5.0 532 L1 C6H5 90 ± 5.0 72.53 L1 o-CH3C6H4 55 ± 4.2 43.84 L1 p-CH3C6H4 90 ± 6.9 71.35 L1 o-OCH3C6H4 31 ± 2.4 23.26 L1 p-OCH3C6H4 60 ± 4.6 15.67 L2 CH3 115 ± 7.0 92.18 L2 C6H5 100 ± 4.9 78.29 L2 o-CH3C6H4 105 ± 6.2 82.510 L2 p-CH3C6H4 105 ± 3.5 84.311 L2 o-OCH3C6H4 104 ± 8.0 83.212 L2 p-OCH3C6H4 102 ± 10.0 80.2

L1, 8-quinoline; L2, 2-methyl-8-quinoline.

direct relationship between the bacterial growth and ureaseinhibitory actions of some cysteine compounds [26].

The nature of PMU inhibition by GeL compoundswas also analyzed by Lineweaver–Burk and Dixon plots(Fig. 3). Inhibition was found to be noncompetitive innature at both acidic (5.6) and basic pH (8.2) values(showed only for compound 5). Similar results were alsoobserved for other GeL compounds (data not shown).GeL compounds decreased Vmax from 40.51% to91.46% without producing an appreciable change in theKm values. The mean Km values were found to bebetween 5.8 and 1.1 lM. Noncompetitive inhibition ofPMU activity suggests either a conformational changeor modification of any functional group of enzyme, at asite other than its active site. This would cause a decreasein Vmax in the presence of inhibitor without any change

yces cerevisiae growth

P. mirabilis S. cerevisiae

M) MIC (lM) L15IC50 (lM)

RS322YIC50 (lM)

106 ± 9.2 ceftriaxone 84 ± 9.4 etoposide —

± 3.0 103 ± 5.0 — —± 2.4 165 ± 4.7 500 500± 2.5 94.3 ± 5.0 500 500± 5.0 175 ± 6.2 500 500± 1.4 61 ± 3.2 500 1000± 1.0 111 ± 5.0 100 250± 1.2 198 ± 10.2 — —± 3.7 195 ± 12.2 500 250± 4.2 180 ± 12.4 250 500± 4.3 182 ± 10.0 250 500± 3.1 185 ± 6.1 250 500± 4.1 199 ± 5.9 250 500

Fig. 3. Lineweaver–Burk plots (left) representing reciprocal of initial PMU velocities versus reciprocal of urea concentrations and Dixon plots (right)representing reciprocal of initial PMU velocities versus GeL compounds concentration in the presence of compound 5 (concentrations mentioned in legendbox) at both pH 5.6 (above) and 8.2 (below). Insets (left): secondary replots of LWB plots i.e. 1/Vmax(app) and slope versus 5 concentrations. Insets (right):secondary replots of the Dixon plot i.e. slope and 1/Vmax(app) of the lines in Dixon plot versus reciprocal of the urea concentrations. Each point representsmean ± SEM (obtained by regression analysis) of three observations (p < 0.05).

Z. Amtul et al. / Biochemical and Biophysical Research Communications 356 (2007) 457–463 461

in the Km values. Noncompetitive inhibition has two sub-types: partial and pure, which can be distinguished on thebasis of replots of slope 1/urea and 1/Vmax(app) versusinhibitor concentration. In the present study the linearplots indicate a pure noncompetitive pattern. As notedearlier, 8-quinolines (L1) derivatives were more potentthan the bulky 2-methyl-8-quinolines (L2) for either anti-bacterial or ureolytic activities (Fig. 2, inset). The impor-tant structural feature for determining the potency of L1over L2 (besides, the bulkiness of L2) was the nature andorientation of R substituent. For instance, replacement ofphenyl group by methyl at R position (compound 2,Table 2) as well as the orientation of electron donatingsubstituents at ortho position (compounds 3 and 5, Table2) of the ring led to an increase in both antibacterial andurease inhibition activities. While comparing the effects ofsubstituents at ortho position it was further observed thatthe ring activation by the methoxy group (donation byconjugation) (compounds 5 and 6, Table 2) resulted ina more pronounced increase in the potency of GeL com-pounds than the activation by methyl substituent (dona-

tion by inductive effect) alone (compounds 3 and 4,Table 2). In 2-methyl-8-quinoline derivatives (L2) the nat-ure of R group did not impart any significant effect ontheir potency, suggesting that primarily the bulkiness ofquinoline group (than the nature of R substituent) wasthe most important structural barrier in increasing theirpotency.

PMU inhibition by GeL compounds was independent ofthe incubation time at the concentrations used. Further-more, the addition of NH2OH (a scavenger of the histidineor tyrosine residue) did not cause any recovery of the ure-ase activity with time (data not shown), implying that his-tidine or tyrosine residues of PMU are not involved in theactivity by these compounds. It is important to note thattwo-pseudo-octahedral paramagnetic nickel ions of ureaseare surrounded by imidazoles of histidine residues [27].This finding further strengthens our above conclusion thatGeL are not binding to the active site of PMU and are non-competitive inhibitors.

In order to evaluate the physiological tolerance of GeLcompounds, various dose levels of the inhibitors were

462 Z. Amtul et al. / Biochemical and Biophysical Research Communications 356 (2007) 457–463

assessed for their mutagenic potential using Saccharomy-

ces cerevisiae strains. None of the inhibitors showed anyconsiderable toxic activity at nuclear level, as they didnot damage the DNA of S. cerevisiae and are not geno-toxic. According to the assay principle, any agent that iscytotoxic (>65% inhibition) to the yeast in the RS322Yplate (mutated species) and not cytotoxic (<35% inhibi-tion) to the yeast in the LF15 plate (wild-type) or alterna-tively an agent whose IC50 values gives a threefolddifferential in the concentration response assay will beconsidered cytotoxic [21]. GeL compounds in generaland L1 derivatives in particular were found to be verymildly toxic against both strains and in a range of 500–1000 lM as compared to the standard ectoposide; a cyto-toxic drug (Table 2).

In summary, our data suggest that the newly synthe-sized GeL compounds are highly selective in inhibitingthe growth of P. mirabilis at moderate concentrations.The inhibition of urease is effective for suppressing thegrowth of P. mirabilis at both basic pH and underacidic conditions. The kinetic data suggests that thesenovel urease inhibitors act in a noncompetitive fashion.Our data indicates that inhibition of urease activity inP. mirabilis may be one mechanism by which GeL com-pounds inhibit their growth; however, it is possible thatthe effect of GeL compounds on P. mirabilis growthmay also be mediated through other mechanisms. Fur-thermore, GeL compounds were not equally potentagainst other urease producing bacteria (for example,Escherichia coli, B. pasteurii and Pseudomonas aerugin-osa) which further suggests that additional mechanismmay be involved in the inhibition of P. mirabilis activityby GeL compounds. The additional molecular targetsfor GeL compounds against P. mirabilis need to beinvestigated.

Acknowledgments

We thank University Grants Commission (UGC) Paki-stan for a predoctoral scholarship to Z.A. and Higher Edu-cation Commission (HEC) Pakistan for the financialassistance under ‘National Research Program forUniversities’.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.bbrc.2007.02.158.

References

[1] L. Pronai, S. Arimori, Protective effect of carboxyethylgermaniumsesquioxide (Ge-132) on superoxide generation by 60Co-irradiatedleukocytes, Biotherapy 3 (3) (1991) 273–279.

[2] Y. Mizushima, Y. Shoji, K. Kaneko, Restoration of impairedimmunoresponse by germanium in mice, Int. Arch. Allergy Appl.Immunol. 63 (3) (1980) 338–339.

[3] M. Hachisu, H. Takahashi, T. Koeda, Y. Sekizawa, Analgesic effectof novel organogermanium compound, GE-132, J. Pharmacobiodyn.6 (11) (1983) 814–820.

\[4] H. Kobayashi, T. Komuro, H. Furue, Effect of combinationimmunochemotherapy with an organogermanium compound, Ge-132, and antitumor agents on C57BL/6 mice bearing Lewis lungcarcinoma (3LL), Gan To Kagaku Ryoho 13 (8) (1986) 2588–2593.

[5] T. Munakata, S. Arai, K. Kuwano, M. Furukawa, Y. Tomita,Induction of interferon production by natural killer cells by organ-ogermanium compound, Ge132, J. Interferon Res. 7 (1) (1987) 69–76.

[6] R.R. Brutkiewicz, F. Suzuki, Biological activities and antitumormechanism of an immunopotentiating organogermanium compound,Ge-132 (Review), In Vivo 1 (1987) 189–203.

[7] C.C. Ho, Y.F. Chern, M.T. Lin, Effects of organogermaniumcompound 2-carboxyethyl germanium sesquioxide on cardiovascularfunction and motor activity in rats, Pharmacology 41 (5) (1990) 286–291.

[8] R.M. Khusainov, M.A. Ignatenko, L.I. Gritsenko, I.S. Frolova, A.A.Babaiants, V.P. Kuznetsov, A.M. Amchenkova, A.N. Narovlianskii,L.N. Pokidysheva, N.S. Barteneva, A new inducer of immuneinterferon in human leukocytes—the organogermanium compoundMOP-11, Vopr. Virusol. 36 (1991) 63–64.

[9] R.E. Mambury, Burger’s Medicinal Chemistry, John Wiley, NewYork, 1979.

[10] B.R. Shavarimuthu, P.T. Muthiah, U. Rychlewska, B. warzajtis, G.Bocelli, R. Olla, Acta Cryst. E59 (2003) m46–m49.

[11] S. Mahboob, M. Parvez, M. Mazhar, S. Ali, Beta-methyl-gamma,-gamma-bis(8-quinolinoxy)germa-gamma-lactone, Acta Crystallo-graphica. Section E E61 (2005) m358–m360.

[12] N. Kakimoto, M. Akiba, T. Takada, Organogermanium compounds,II. A simple synthesis of substituted 3-trichlorogermylpropanoic acidsand esters by regioselective hydrogermylation, Synthesis (1985) 272–274.

[13] S. Li-Juan, W. Yu-Sheng, B. Ming-Zhang, Synthesis of 3-substitutedphenyl-3-trichlorogermyl propionic acids, Youji Huaxue 11 (1991)303–306.

[14] S. Xueqing, Y. Zhiqiang, X. Qinglan, L. Jinshan, Synthesis, structureand in vitro antitumor activity of some germanium-substituted di-N-butyl dipropionates, J. Organomet. Chem. 566 (1988) 103–110.

[15] G.Q. Shangguan, S.G. Zhang, J. Zuan, Ni synthesis, structure andantitumor activities of—phenolester propyl germanium sesquioxides,Chinese Chem. Lett. 6 (11) (1995) 945–946.

[16] M.A. Choudhary, M. Mazhar, U. Salma, S. Ali, X. Qinglan, K.C.Molloy, Synthesis and characterization of diorganotin compoundscontaining silicon and germanium and crystal structure of precursorcarboxyethyltrichlorogermanium, Synth. React. Inorg. Met. Org.Chem. 31 (2) (2001) 277–295.

[17] P.R. Murray, E.J. Baron, J.H. Jorgensen, et al., Manual of ClinicalMicrobiology, ASM Press, Washington, DC, 2003.

[18] J.M. Breitenbach, R.P. Hausinger, Proteus mirabilis urease. Partialpurification and inhibition by boric acid and boronic acids, Biochem.J. 250 (3) (1988) 917–920.

[19] J.T. Bosse, J.I. MacInnes, Genetic and biochemical analyses ofActinobacillus pleuropneumoniae urease, Infect. Immun. 65 (11) (1997)4389–4394.

[20] M.W. Weatherburn, Phenol-hypochlorite reaction for determinationof ammonia, Anal. Chem. 39 (1967) 971–974.

[21] V. Afanassiev, M. Sefton, T. Anantachaiyong, G. Barker, R.Walmsley, S. Wolfl, Application of yeast cells transformed withGFP expression constructs containing the RAD54 or RNR2 pro-moter as a test for the genotoxic potential of chemical substances,Mutat. Res. 464 (2) (2000) 297–308.

[22] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Proteinmeasurement with the Folin phenol reagent, J. Biol. Chem. 193 (1)(1951) 265–275.

[23] S. Goodman, Therapeutic effects of organic germanium, Med.Hypotheses 26 (3) (1988) 207–215.

Z. Amtul et al. / Biochemical and Biophysical Research Communications 356 (2007) 457–463 463

[24] D.E. Johnson, R.G. Russell, C.V. Lockatell, J.C. Zulty, J.W.Warren, H.L. Mobley, Contribution of Proteus mirabilis urease topersistence, urolithiasis, and acute pyelonephritis in a mouse modelof ascending urinary tract infection, Infect. Immun. 61 (7) (1993)2748–2754.

[25] X. Li, C.V. Lockatell, D.E. Johnson, H.L. Mobley, Identification ofMrpI as the sole recombinase that regulates the phase variation ofMR/P fimbria, a bladder colonization factor of uropathogenicProteus mirabilis, Mol. Microbiol. 45 (3) (2002) 865–874.

[26] Z. Amtul, N. Kausar, C. Follmer, R.F. Rozmahel, R. Atta Ur, S.A.Kazmi, M.S. Shekhani, J.L. Eriksen, K.M. Khan, M.I. Choudhary,Cysteine based novel noncompetitive inhibitors of urease(s)—distinc-tive inhibition susceptibility of microbial and plant ureases, Bioorg.Med. Chem. 14 (19) (2006) 6737–6744.

[27] N.E. Dixon, R.L. Blakeley, B. Zerner, Jack bean urease (EC 3.5.1.5).III. The involvement of active-site nickel ion in inhibition by beta-mercaptoethanol, phosphoramidate, and fluoride, Can J. Biochem. 58(6) (1980) 481–488.