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In vitro transcription of capped mRNA with modified nucleotides and Poly(A) tail
TSA (Tyramide Signal Amplification), used for signal amplification of ISH, IHC and IC etc.
Separation of phosphorylated and non-phosphorylated proteins without phospho-specific antibody
A convenient and sensitive way for cell proliferation assay and cytotoxicity assay
Protect the integrity of proteins from multiple proteases and phosphatases for different applications.
CHIR-090 is a very potent, low, tight-binding inhibitor of LpxC with Ki value of 4.0 nM [1].LpxC is a zinc-dependent amidase and present in almost all Gram-negative bacteria. LpxC is a promising target for the development of novel antibiotic substances against multigrug-resistant Gram-negative bacteria [2].CHIR-090 is a potent LpxC inhibitor and has a different selectivity with the reported LpxC inhibitor L-161. When tested with Escherichia coli LpxC, administration of CHIR-090 showed tight inhibition with Ki value of 4.0 nM, Ki*=0.5 nM, K5=1.9/min and K6=0.18/min [1]. In bacterial P.aeruginosa efflux pupm mutants, CHIR-090 treatment showed inhibition function on MexAB-Oprm, MexCD-OprJ and MexEF-OprN [2]. CHIR-090 showed remarkable antibiotic activity against both E.coli and P.aeruginosa by inhibiting LpxC orthologs at low nM concentrations [3].In E.coli W3110RL with R.legumunosarum lpxC replacement of E.coli lpxC, CHIR-090 (1 to 10 μg/ml) treatment had no effect on strain growth on LB agar plates while wild-type cells stopped growing after about 2 h in the presence of 1 μg/ml CHIR-090 [1].References: [1].Barb, A.W., et al., Inhibition of lipid A biosynthesis as the primary mechanism of CHIR-090 antibiotic activity in Escherichia coli. Biochemistry, 2007. 46(12): p. 3793-802.[2].Barb, A.W. and P. Zhou, Mechanism and inhibition of LpxC: an essential zinc-dependent deacetylase of bacterial lipid A synthesis. Curr Pharm Biotechnol, 2008. 9(1): p. 9-15.[3].McClerren, A.L., et al., A slow, tight-binding inhibitor of the zinc-dependent deacetylase LpxC of lipid A biosynthesis with antibiotic activity comparable to ciprofloxacin. Biochemistry, 2005. 44(50): p. 16574-83.
Assay of LpxC Activity
UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine and [α-32P] UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine were prepared enzymatically as previously described. Assays of LpxC activity were performed with 5 μM substrate, except where noted; additionally, 10% DMSO was added to the assay mixtures and held constant at that level when inhibitor (dissolved in DMSO) was added. Except where noted, the concentration of the enzyme was at least 10-fold less than the concentration of either the inhibitor or the substrate. When pre-incubated with or without inhibitor prior to being assayed, the enzyme was diluted in 25 mM sodium phosphate, pH 7.4, containing 1 mg/mL BSA and 10% DMSO. The pre-incubation mixture was held on ice for 15 min before the reaction was initiated by means of a 1:4 dilution of the enzyme into the assay cocktail. Initial velocities were calculated from the linear portion of reaction progress curves (
Cell lines
wild-type E. coli W3110 and E. coli W3110RL
Preparation method
The solubility of this compound in DMSO is >21.9mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.
Reacting condition
1 μg/mL
Applications
In the presence of 1 μg/mL CHIR-090, wild-type E. coli W3110 stopped growing after about 2 h. The E. coli W3110RL, in which R. leguminosarum lpxC replaces the chromosomal copy of E. coli lpxC, was resistant to CHIR-090. The MIC of CHIR-090 against W3110RL in liquid medium was 100 μg/mL, compared to 0.25 μg/ mL for W3110.
References:
[1].Barb, A.W., et al., Inhibition of lipid A biosynthesis as the primary mechanism of CHIR-090 antibiotic activity in Escherichia coli. Biochemistry, 2007. 46(12): p. 3793-802.