In Vitro Validation of Acetyltransferase Activity of GlmU as an Antibacterial Target in Haemophilus influenzae

A series of inhibitors with a squaramide core was synthesized following its discovery in a high-throughput screen for novel inhibitors of a transcription-coupled translation assay using Escherichia coli S30 extracts. The inhibitors were inactive when the plasmid substrate was replaced with mRNA, suggesting they interfered with transcription. This was confirmed by their inhibition of purified E. coli RNA polymerase. The series had antimicrobial activity against efflux-negative strains of E. coli and Haemophilus influenzae. Like rifampin, the squaramides preferentially inhibited synthesis of RNA and protein over fatty acids, peptidoglycan, and DNA. However, squaramide-resistant mutants were not cross-resistant to rifampin. Nine different mutations were found in parts of rpoB or rpoC that together encode the so-called switch region of RNA polymerase. This is the binding site of the natural antibiotics myxopyronin, corallopyronin, and ripostatin and the drug fidaxomicin. Computational modeling using the X-ray crystal structure of the myxopyronin-bound RNA polymerase of Thermus thermophilus suggests a binding mode of these inhibitors that is consistent with the resistance mutations. The squaramides are the first reported non-natural-productrelated, rapidly diversifiable antibacterial inhibitors acting via the switch region of RNA polymerase. C linical resistance to currently prescribed antibiotics is on the rise, thus increasing the need for new classes of antimicrobials that can circumvent emerging resistance mechanisms (10). There are still only a few enzymes that are essential for bacterial growth and have been clinically validated as antibacterial targets. All clinical antibacterial protein translation inhibitors have so far been identified by cell-based screening efforts with compounds from natural sources (8). New, small inhibitors might be found by screening small-molecule libraries for inhibitors of the translation machinery with an in vitro system, such as transcription-coupled translation in bacterial S30 extracts.Here, we report the discovery of squaramides as inhibitors of RNA polymerase (RNAP) that resulted from such a screening effort. The antimicrobial activity against an efflux-negative strain of H. influenzae was exploited to show that squaramides mediate their inhibitory activity via the switch region of RNAP. Their mode of action therefore is similar to that of the natural compounds myxopyronin, corallopyronin, ripostatin, and fidaxomicin (26) rather than that of rifamycins, which bind closer to the catalytic site and prevent RNA extension (7). This is the first report of rapidly diversifiable small-molecule inhibitors of RNAP with that mode of inhibition, supporting the use of a transcription-coupled translation assay to find novel inhibitory scaffolds of the RNAP switch region in small-molecule collections. MATERIALS AND METHODSBacterial strains. E. coli RNAP and S30 extracts were isolated from E. coli MRE600 (ATCC 2941). For susceptibility studies E. coli ATCC 27325 and H. influenzae ATCC 51907 ...

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“…The procedure was essentially that according to Hilliard et al (14), with modifications published previously (5).…”

Section: Methodsmentioning

confidence: 99%

Novel Rapidly Diversifiable Antimicrobial RNA Polymerase Switch Region Inhibitors with Confirmed Mode of Action in Haemophilus influenzae

et al. 2012

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“…Zhang et al (8), by modeling acetyl-CoA into the apo structure of GlmU Mtb , suggested an interaction of Trp-460 and the substrate. Crystal structures of GlmU Hi (from Haemophilus influenzae) bound with acetyltransferase inhibitors were reported recently (21). These depict an important contribution by the equivalent Trp-449 in inhibitor binding.…”

Section: Ecmentioning

confidence: 99%

Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

Jagtap

Soni

Vithani

et al. 2012

Journal of Biological Chemistry

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Background: Catalytic mechanism for acetyltransferase activity and its regulation in M. tuberculosis GlmU (GlmU Mtb ) is not addressed comprehensively. Results: We present these and features unique to GlmU Mtb . Conclusion: Unique features include a short helix that presents Trp-460 for substrate binding, distinctive acetyl-CoA conformation and regulation upon PknB-mediated phosphorylation. Significance: These insights may be exploited for designing selective inhibitors against GlmUMtb .

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“…3,5 In addition, the acetyltransferase domain of GlmU has also been validated in vitro as an antibacterial target in Haemophilus influenzae, making it a suitable target for drug development. 6 The structure of GlmU enzyme and its mechanism has been revealed from the crystallographic studies of the GlmU enzyme with its substrates and inhibitors. The N-terminal domain (residues Asn3-Arg229) adopts an α/β fold similar to the dinucleotide binding Rossmann fold, whereas the C-terminal domain (residues Phe260-Ala437) catalyzes the acetyltransferase activity and adopts a left-handed parallel β-helix (LβH) structure homologous to bacterial acetyltransferases (Suppl.…”

Section: Introductionmentioning

confidence: 99%

“…10 Multiple screening strategies also led to the discovery of novel E. coli GlmU inhibitors, including arylamines 11 and aryl sulfonamides. 6,12,13 Recently, an in silico screening strategy using ligand-guided and structure-guided techniques led to the identification of two promising sulfonamide inhibitors. 14 Terreic acid is a reported antibacterial inhibitor, but its cellular and molecular targets are still unknown.…”

Section: Introductionmentioning

confidence: 99%

Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus

et al. 2016

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Secondary metabolite of Aspergillus terreus, terreic acid, is a reported potent antibacterial that was identified more than 60 years ago, but its cellular target(s) are still unknown. Here we screen its activity against the acetyltransferase domain of a bifunctional enzyme, Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). An absorbance-based assay was used to screen terreic acid against the acetyltransferase activity of E. coli GlmU. Terreic acid was found to inhibit the acetyltransferase domain of E. coli GlmU with an IC 50 of 44.24 ± 1.85 µM. Mode of inhibition studies revealed that terreic acid was competitive with AcCoA and uncompetitive with GlcN-1-P. It also exhibited concentration-dependent killing of E. coli ATCC 25922 up to 4× minimum inhibitory concentration and inhibited the growth of biofilms generated by E. coli. Characterization of resistant mutants established mutation in the acetyltransferase domain of GlmU. Terreic acid was also found to be metabolically stable in the in vitro incubations with rat liver microsome in the presence of a NADPH regenerating system. The studies reported here suggest that terreic acid is a potent antimicrobial agent and support that E. coli GlmU acetyltransferase is a molecular target of terreic acid, resulting in its antibacterial activity.

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In Vitro Validation of Acetyltransferase Activity of GlmU as an Antibacterial Target in Haemophilus influenzae

Cited by 37 publications

References 22 publications

Novel Rapidly Diversifiable Antimicrobial RNA Polymerase Switch Region Inhibitors with Confirmed Mode of Action in Haemophilus influenzae

Novel Rapidly Diversifiable Antimicrobial RNA Polymerase Switch Region Inhibitors with Confirmed Mode of Action in Haemophilus influenzae

Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus

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