Selective Methyl Labeling of Proteins: Enabling Structural and Mechanistic Studies As Well As Drug Discovery Applications by Solution-State NMR

Proudfoot, Andrew; Frank, Albin; Frommlet, Alexandra; Lingel, Andreas

doi:10.1016/bs.mie.2018.08.035

Cited by 9 publications

(13 citation statements)

References 60 publications

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“…The LpxA SPR binding assay was performed using Biacore T200. Protein-observed NMR using isotope-enriched LpxA-His6 protein was performed as described previously . The X-ray crystal structures of the LpxA/ligand complexes were obtained by crystal soaking and solved by molecular replacement.…”

Section: General Methodsmentioning

confidence: 99%

Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis

Han

Balibar

et al. 2020

J. Am. Chem. Soc.

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The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

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Section: General Methodsmentioning

confidence: 99%

Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis

Han

Balibar

et al. 2020

J. Am. Chem. Soc.

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“…Based on the unfavorable forward reaction of LpxA 23 and the stable enzyme/product complex demonstrated by the crystal structure with UDP-3- O -( R -3-hydroxymyristoyl)-GlcNAc 28 , we postulated that compound 2 might bind the LpxA-product complex. To examine the binding mechanism, we developed a two-dimensional protein-observed HMQC NMR assay 43–44 for E. coli LpxA, using selective incorporation of 1 H, 13 C isotope labels at all methyl positions of CH 3 -containing amino acids (Met, Ile, Leu, Val, Ala, and Thr, but not Ile Cγ). As expected, robust chemical shift perturbations (CSP) were observed when isotope-labeled LpxA was incubated with compound 1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Protein-observed NMR using isotope-enriched LpxA-His6 protein was performed as described previously. 43 The X-ray crystal structures of the LpxA-ligand complexes were obtained by crystal soaking and solved by molecular replacement. Compound 1 and 5-(4-methylpyridin-2-yl)-1H-pyrazole-3-carboxylic acid for compound 2 were prepared using literature procedures.…”

Section: Methodsmentioning

confidence: 99%

Two distinct mechanisms of small molecule inhibition of LpxA acyltransferase essential for lipopolysaccharide biosynthesis

Han¹,

Ma²,

Balibar³

et al. 2019

Preprint

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“…For NMR experiments, uniformly 15 N/ 2 H labeled LxpA-His6 protein with selectively introduced 13 C methyl groups was prepared. To maximize the number of NMR-active probes, we simultaneously labeled the methyl groups of all methyl containing amino acids (methionine, isoleucine, leucine, valine, alanine, threonine, known as MILVAT labeling scheme) according to the recently published protocol [6][7] . Purification of isotope labeled LpxA-His6 protein was performed as described above for unlabeled LpxA-His6 protein, except that buffer comprised solely of 50 mM sodium phosphate monobasic pH 7.0 was used.…”

Section: Preparation Of Isotope-enriched Lpxa-his6 Protein For Nmr Stmentioning

confidence: 99%

“…Samples contained 80 µM isotope-labeled (MILVAT methyl labeled) LpxA, and 100 µM compound 1 or compound 2 with 150 µM UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc (Toronto Research Chemicals) in 50 mM sodium phosphate buffer pH 7.0, 100 mM sodium chloride, 2 mM deuterated DTT, and 10 µM DSS. Two-dimensional 13 C-1 H SOFAST HMQC spectra [6][7]13 were recorded with 64 scans and up to 96 points in the indirect dimension.…”

Section: Protein Observed 2d Nmr Assaymentioning

confidence: 99%

Two distinct mechanisms of small molecule inhibition of LpxA acyltransferase essential for lipopolysaccharide biosynthesis

Han

Balibar

et al. 2019

Preprint

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This PDF file includes:Supplementary Methods Figs. S1 to S5 Tables S1 to S3 Supplementary Information Text Bacterial strains, plasmids, routine growth condition, and compound susceptibility testBacterial strains and plasmids used in this study are listed in Table S1. Miller's lysogeny broth (LB) 1 and LB-agar were used for routine growth and bacterial selection during plasmid construction. To maintain plasmids, 100 µg/mL ampicillin (Ap), 10 µg/mL chloramphenicol (Cm), and 50 µg/mL kanamycin (Km) were added to the media as needed. Compound susceptibility testing to determine MICs was performed in cation adjusted Mueller-Hinton II broth (CAMHB) according to CLSI protocols, as described previously 2 . To induce genes under the control of a lac promoter, 100 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the media used in susceptibility testing. Compounds were synthesized at Novartis as described below. CHIR-090 was purchased from ChemScene (CS-0973). Spontaneous mutant selectionFor mutant selection with compound 1, cells in overnight culture of MG1655 ΔtolC were spread on LB agar containing 0.5 µg/mL compound 1. For mutant selection with compound 14, overnight culture of CDY0154 was used and the cells were spread on L-agar containing 16 and 32 µg/mL compound 14. Serial dilutions of the overnight culture were spread on LB agar to determine the number of cells (approximately 10 8 and 10 9 cells) used for selection. The LB agar plates were incubated overnight at 37 °C. Colonies arose and were streaked on fresh LB agar. After incubation overnight at 37 °C, a single colony of each mutant was grown in LB and stored in 15% glycerol at -80 °C for genome sequencing and susceptibility tests. The fabZ mutants were isolated from selection with inhibitors of LpxA and LpxD. Genome sequencing and mutation confirmationGenomic DNA for whole genome sequencing was isolated from E. coli strains using the Qiagen DNeasy Blood and Tissue kit with following the instruction for Gram-negative bacterial DNA. To sequence the genomes of isolated mutants, library preparation and sequencing reaction for next generation sequencing, and data analysis were performed using the Illumina Sample Preparation kits and Illumina Sequencer as described previously 2 . Sequencing reads were aligned to the reference E. coli MG1655 genome (Genbank ID: NC_000913.3) using bwa for determining single nucleotide polymorphisms and short insertion/deletions (indels, typically <10 bp). Mutations identified by genomics were confirmed by targeted PCR and Sanger sequencing. The primers were used to amplify the gene of interest with Phusion High-Fidelity PCR Master Mix with GC Buffer according to established protocols. Sequencing was performed by Elim BioPharma (Hayward, CA). The primers used for PCR and sequencing were TU117, TU243, TU276, TU332, TU475, TU503, TU504, TU512, and TU513. Construction of CDY0154The acrAB locus was replaced with the kanamycin resistant cassette (Km r ) using the λRED recombination system as described previously 3 . The DNA fragmen...

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Selective Methyl Labeling of Proteins: Enabling Structural and Mechanistic Studies As Well As Drug Discovery Applications by Solution-State NMR

Cited by 9 publications

References 60 publications

Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis

Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis

Two distinct mechanisms of small molecule inhibition of LpxA acyltransferase essential for lipopolysaccharide biosynthesis

Two distinct mechanisms of small molecule inhibition of LpxA acyltransferase essential for lipopolysaccharide biosynthesis

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