A kinetic mechanism for the poly(C)-dependent ATPase of the Escherichia coli transcription termination protein, rho.

Sharp, J A; Galloway, Jenna L.; Platt, Terry

doi:10.1016/s0021-9258(18)32687-5

Cited by 39 publications

(58 citation statements)

References 18 publications

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“… a Activity measured using the ATPase assay (ref ) and described in an earlier paper (ref ). b The I 50 value is the average 50% inhibition concentration determined from duplicate tests.…”

Section: Resultsmentioning

confidence: 99%

Design, Syntheses, and Evaluations of Bicyclomycin-Based Rho Inactivators

et al. 1997

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The commercial antibiotic bicyclomycin (1) has been shown to target the essential transcriptional termination factor rho in Escherichia coli. Little is known, however, about the bicyclomycin binding site in rho. A recent structure−activity relationship study permitted us to design modified bicyclomycins that may irreversibly inactivate rho. The four compounds selected were C(5a)-(4-azidoanilino)dihydrobicyclomycin (3), C(5a)-(3-formylanilino)dihydrobicyclomycin (4), C(5)−norbicyclomycin C(5)-O-(4-azidobenzoate) (5), and C(5)-norbicyclomycin C(5)-O-(3-formylbenzoate) (6). In each of these compounds the inactivating unit was placed at the C(5)−C(5a) site in bicyclomycin. In compounds 3 and 5 an aryl azide moiety was used as a photoaffinity label whereas in 4 and 6 an aryl aldehyde group was employed as a reductive amination probe. The synthesis and spectral properties of 3−6 are described. Chemical studies demonstrated that 3 and 4 were stable in D2O and CD3OD (room temperature, 7 d), while 5 and 6 underwent significant change within 1 d. Biochemical investigations showed that 3 and 4 retained appreciable inhibitory activities in rho-dependent ATPase and transcription termination assays. In the ATPase assay, I 50 values for 1, 3, and 4 were 60, 135, and 70 μM, respectively. Correspondingly, the I 50 values for 5 and 6 were >400 and 225 μM, respectively. In the transcription termination assay, compounds 1, 3, and 4 all prevented (≥97%) the production of rho-dependent transcripts at 40 μM, whereas little (≤15%) inhibition of transcription termination was observed for 5 and 6 at this concentration. Antimicrobial evaluation of 3−6 showed that none of the four compounds exhibited antibiotic activity at 32 mg/mL or less against W3350 E. coli. The combined chemical and biochemical studies led to our further evaluation of 3 and 4. Photochemical irradiation (254 nm) of 3 in the presence of rho led to a 29−32% loss of rho ATPase activity. Attempts to confirm the irreversible adduction of 3 to rho by electrospray mass spectrometry were unsuccessful. No higher molecular weight adducts were detected. Incubation of rho with 4 at room temperature (4 h) followed by the addition of NaBH4 led to significant losses (>62%) of rho ATPase activity. Analyses of the 4−rho modified adduct showed appreciable levels of adduction (∼40%). Mass spectrometric analyses indicated a molecular weight for the adduct of approximately 47 410, consistent with a modification of a rho lysine residue by 4. Compound 4 was selected for additional studies.

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“… a Activity measured using the ATPase assay (ref ) and described in an earlier paper (ref ). b The I 50 value is the average 50% inhibition concentration determined from duplicate tests.…”

Section: Resultsmentioning

confidence: 99%

Design, Syntheses, and Evaluations of Bicyclomycin-Based Rho Inactivators

et al. 1997

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“…The first describes the C(5)-unsaturated adducts 5 − 14 . The inhibitory properties of these compounds in the poly(C)-dependent rho ATPase assay are listed in Table A. Most of the compounds effectively inhibited (56−90%) ATP hydrolysis at 400 μM.…”

Section: Resultsmentioning

confidence: 99%

“…General Methods. Procedures identical to those previously described 24 were used in the synthesis and evaluation of bicyclomycin C(5)−C(5a) exomethylene-modified derivatives in the poly(C)-dependent ATPase, rho-dependent transcription termination, and antimicrobial assays…”

Section: Methodsmentioning

confidence: 99%

Role of the C(5)−C(5a) Exomethylene Group in Bicyclomycin: Synthesis, Structure, and Biochemical and Biological Properties

Park

Zhang

et al. 1996

J. Org. Chem.

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Thirty-two C(5)-C(5a) exomethylene-modified bicyclomycin derivatives were prepared to determine the effect of structural modification of this unit on bicyclomycin (1) function. The compounds were grouped into three categories: the C(5)-unsaturated bicyclomycins, the C(5a)-substituted C(5)-C(5a)-dihydrobicyclomycin derivatives, and the C(5)-modified norbicyclomycins. An efficient three-step procedure was developed to synthesize C(5a)-substituted C(5),C(5a)-dihydrobicyclomycins. Bicyclomycin was converted to bicyclomycin C(2'),C(3')-acetonide (36) and then treated with a nucleophile in 50% aqueous methanol ("pH" 10.5) to give the C(5a)-substituted C(5),C(5a)-dihydrobicyclomycin C(2'),C(3')-acetonide. Removal of the acetonide group (trifluoroacetic acid in 50% aqueous methanol) in the final step provided the desired bicyclomycin derivative. All the compounds were evaluated using the rho-dependent ATPase assay and their antimicrobial activities determined using a filter disc assay. Most of the compounds were also tested in the transcription termination assay. We observed that many of the C(5)-unsaturated bicyclomycins effectively inhibited ATP hydrolysis at 400 &mgr;M and inhibited the production of rho-dependent transcripts at 100 &mgr;M. The biochemical activities of C(5a)-bicyclomycincarboxylic acid (5), methyl C(5a)-bicyclomycincarboxylate (6), ethyl C(5a)-bicyclomycincarboxylate (7), and bicyclomycin C(5)-norketone O-methyloxime (11) were all similar to 1. Compounds 6, 7, and 11 exhibited diminished antibiotic activity compared to 1, and 5 displayed no detectable activity. Several C(5a)-substituted C(5),C(5a)-dihydrobicyclomycins showed significant inhibition of rho-dependent ATPase and transcription termination activities. The inhibitory properties of C(5),C(5a)-dihydrobicyclomycin C(5a)-methyl sulfide (18), C(5),C(5a)-dihydrobicyclomycin C(5a)-phenyl sulfide (23), and C(5)-C(5a)-dihydrobicyclomycin-5,5a-diol (31) approached those of 1. Compounds 18, 23, and 31 did not exhibit antibiotic activity. Two of the four C(5)-modified norbicyclomycin adducts showed moderate inhibitory activities in the ATPase assay, and none showed significant antibiotic activity. Our findings showed that the C(5)-C(5a) exomethylene unit retention in 1 was not essential for inhibition of in vitro rho activity. The structure-activity relationship data indicated that bicyclomycins that contained a small unsaturated C(5) unit or C(5),C(5a)-dihydrobicyclomycins that possessed a small, nonpolar C(5a) substituent effectively inhibited rho function in in vitro biochemical assays. We concluded that the C(5)-C(5a) unit in 1 was not a critical structural element necessary for drug binding to rho and that irreversible, inactivating units placed at this site would permit the bicyclomycin derivative to bind efficiently to rho.

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“…Table 3 shows that successive N-methylation of the piperazinedione ring led to lower inhibitory activities in the rho ATPase assay. 18 For example the I 50 values of 1 19 and 2 were 60 and 350 µM, respectively. Similarly, N(8),N(10)-dimethylbicyclomycin (3) showed less than 5% inhibition of ATPase activity at a 400 µM concentration while bicyclomycin (1) inhibited ATP hydrolysis by 95% at this concentration.…”

Section: E Biochemical and Biologicalmentioning

confidence: 99%

Role of the [4.2.2] Bicyclic Unit in Bicyclomycin: Synthesis, Structure, Chemical, Biochemical, and Biological Properties

et al. 1996

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Twelve bicyclomycin derivatives were synthesized to determine the effect of modification of the [4.2.2] bicyclic unit in bicyclomycin (1) on drug function. Few bicyclomycin derivatives have been described in which the [4.2.2] ring system has been modified. The compounds evaluated were divided into two categories: the two N-methyl-modified bicyclomycins (2, 3) and the ten C(6)-substituted bicyclomycins (4-13). Substituents introduced at the C(6) site included alkoxy, thioalkoxy, thiophenoxy, anilino, and hydrogen. A procedure was developed to synthesize select C(6)-substituted bicyclomycins. Bicyclomycin was first converted to bicyclomycin C(2'),C(3')-acetonide (16) and then treated with methanesulfonyl chloride to give in situ the corresponding C(6) mesylate 17. Treatment of 17 with the appropriate nucleophile followed by removal of the C(2'),C(3')-acetonide group gave the desired C(6)-substituted bicyclomycin. The chemical properties of C(6) O-methylbicyclomycin (4) were examined. Treatment of THF-H(2)O mixtures of 4 with excess EtSH maintained at "pH" 8.0-9.0 led to no detectable reaction, while at more basic "pH" values 4 underwent stereospecific conversion to the bis-spiro derivative 33 and no appreciable EtSH addition to the C(5)-C(5a) exomethylene unit. These results were compared to the reactivity of 1 with EtSH. The stability (pH 7.4, 37 degrees C) of C(6)-substituted bicyclomycins 4, 6, and 10-13 in aqueous solutions were examined. We observed that most of these compounds (4, 6, 10-12) underwent near complete change (>75%) within 200 h. The [4.2.2] bicyclic-modified bicyclomycins were evaluated in the rho-dependent ATPase assay and their antimicrobial activities determined using a filter disc assay. Most of the compounds were also tested in the transcription termination assay. We observed that all structural modifications conducted within the [4.2.2] bicyclic unit led to a loss of rho-dependent ATPase (I(50) > 400 &mgr;M) and to transcription termination (I(50) > 100 &mgr;M) inhibitory activities, as well as a loss of antimicrobial activity (MIC > 32 mg/mL). Only N(10)-methylbicyclomycin (2) displayed moderate inhibitory activities in these assays. These findings indicated that the [4.2.2] bicyclic unit played an important role in the antibiotic-rho recognition process. Potential factors that govern this interaction are briefly discussed. We concluded that placement of an irreversible inactivating unit at the N- and O-sites within the [4.2.2] bicyclic unit in 1 would likely prohibit the bicyclomycin derivative from efficiently binding to rho.

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A kinetic mechanism for the poly(C)-dependent ATPase of the Escherichia coli transcription termination protein, rho.

Cited by 39 publications

References 18 publications

Design, Syntheses, and Evaluations of Bicyclomycin-Based Rho Inactivators

Design, Syntheses, and Evaluations of Bicyclomycin-Based Rho Inactivators

Role of the C(5)−C(5a) Exomethylene Group in Bicyclomycin: Synthesis, Structure, and Biochemical and Biological Properties

Role of the [4.2.2] Bicyclic Unit in Bicyclomycin: Synthesis, Structure, Chemical, Biochemical, and Biological Properties

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