Inhibition of Pseudomonas aeruginosa LpxC expressed in Escherichia coli using UDP-3-O-N-acetylglucosamine substrate measured after 30 mins by mass spectrometry
Inhibition of Pseudomonas aeruginosa LpxC using UDP-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine incubated for 30 mins prior to substrate addition measured after 60 mins by mass spectrophotometry
Inhibition of Pseudomonas aeruginosa LpxC using UDP-3-O-(R-3-hydroxydecanoyl)-N-acetylglucosamine as substrate preincubated for 20 mins before substrate addition by LC/MS/MS analysis
Enzyme Assay: IC50 determination in the LpxC enzyme assay was carried out in a similar manner to that described by Malikzay et al in the 2006 Poster, Screening LpxC (UDP-3-O(R-3-hydroxymyristoyl)-GlcNAc deacetylase) using BioTrove Rapid Fire HTS Mass Spectrometry (aNew Lead Discovery and bInflammation and Infectious Disease, cStructural Chemistry, Schering-Plough Research Institute, Kenilworth, N.J. 07033, (BioTrove, Inc. 12 Gill St., Suite 4000, Woburn, Mass. 01801). Briefly, Pseudomonas aeruginosa LpxC enzyme (0.1 nM) purified from E. coli-overexpressing bacteria was incubated at 25° C. in a final volume of 50 ul containing 0.5 uM UDP-3-O(R-3-hydroxydecanoyl)-N-acetylglucosamine, 1 mg/mL BSA, and 50 mM sodium phosphate buffer, pH 8.0 in the presence and absence of inhibitor compound. At the end of 1 hour, 5 ul of 1N HCl was added to stop the enzyme reaction; the plates were centrifuged, and then processed with the BioTrove Rapidfire HTMS Mass Spectrometry System.
Inhibition Assay: To assay the activity of Pseudomonas aeruginosa LpxC enzyme, LpxC was reacted with its substrate UDP-3-O-(R-3-hydroxydecanoyl)-N-acetylglucosamine and the amount of the reaction product was determined by quantifying the amino groups present in it.Specifically, 12.5 ng of Pseudomonas aeruginosa LpxC enzyme (as acquired by preparing chromosomal DNA from Pseudomonas aeruginosa, subjecting the DNA to PCR (polymerase chain reaction) using LpxC specific primers to acquire Pseudomonas aeruginosa LpxC genes, incorporating the genes into a vector, and expressing the same with E. coli) was mixed with 80 .mu.mol/L of UDP-3-O-(R-3-hydroxydecanoyl)-N-acetylglucosamine (Wako Pure Chemical Industries, Ltd.) and the mixture was incubated at room temperature for 40 minutes. The reaction was performed in 40 mmol/L of Hepes buffer solution (pH 8.0) supplemented with 0.02% Bridge 35 and 80 .mu.mol/L of dithiothreitol. To terminate the reaction, 0.2 mol/L of borax was added to the reaction mixture.
Inhibition of wild-type Pseudomonas aeruginosa LpxC preincubated for 20 mins followed by addition of UDP-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine as substrate measured after 60 mins by HPLC analysis
Inhibition of wild-type Pseudomonas aeruginosa PAO1 LpxC assessed as deacetylation preincubated for 30 mins followed by addition of UDP-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine as substrate measured after 60 mins by mass spectrometry
Inhibition of Pseudomonas aeruginosa LpxC using UDP-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine as substrate preincubated for 30 mins followed by substrate addition measured after 20 mins by LC-MS/MS analysis
Binding affinity to recombinant Pseudomonas aeruginosa LpxC C40S mutant (1 to 299 residues) expressed in Escherichia coli BL21 AI cells assessed as change in melting temperature at 25 uM SYPRO orange dye based differential scanning fluorimetric method
Inhibition of recombinant Pseudomonas aeruginosa LpxC (1 to 299 residues) C40S mutant expressed in Escherichia coli BL21 AI assessed as change in melting temperature at 25 uM by Sypro orange dye based differential scanning fluorimetry
Inhibition of LpxC in Pseudomonas aeruginosa assessed as reduction in bacterial growth measured after 20 hrs by CLSI protocol based microbroth dilution method
Inhibition of LpxC in Pseudomonas aeruginosa assessed as delay in bacterial growth at 25 to 50 ug/ml measured after 20 hrs by CLSI protocol based microbroth dilution method
Inhibition of histidine-tagged Pseudomonas aeruginosa LpxC (1-303) expressed in Escherichia coli BL21 (DE3) using UDP-3-O-(R-3-hydroxydecanoyl)-N-acetylglucosamine as substrate measured after 60 mins by Fluorescence plate reader analysis