Overexpression of Pseudomonas aeruginosa LpxC with its inhibitors in an acrB-deficient Escherichia coli strain

Gao, Ning; McLeod, Sarah M.; Hajec, Laurel I.; Olivier, N.B.; Lahiri, S. C.; Prince, D. Bryan; Thresher, Jason; Ross, Philip L.; Whiteaker, James; Doig, P.; Li, Amanda Haixi; Hill, Pamela; Cornebise, Mark; Reck, Folkert; Hale, Michael R.

doi:10.1016/j.pep.2014.09.006

Cited by 6 publications

(2 citation statements)

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“…Compound 4 48 and Compound 5 (example 37 49 ) were prepared as described previously and Compound 6 (PF-5081090) was acquired from Sigma-Aldrich (Cat. number PZ0194).…”

Section: Methodsmentioning

confidence: 99%

Translating slow-binding inhibition kinetics into cellular and in vivo effects

et al. 2015

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Many drug candidates fail in clinical trials due to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug:target complex has a longer half-life than off-target:drug complexes. However, while slow-binding inhibition kinetics are a key feature of many marketed drugs1,2, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.

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“…Compound 4 48 and Compound 5 (example 37 49 ) were prepared as described previously and Compound 6 (PF-5081090) was acquired from Sigma-Aldrich (Cat. number PZ0194).…”

Section: Methodsmentioning

confidence: 99%

Translating slow-binding inhibition kinetics into cellular and in vivo effects

et al. 2015

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“…The published crystal structures of the LpxA and LpxC from E. coli contain UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine and its deacetylated product, respectively [ 16 , 17 ]. These proteins catalyze the first committed step of lipid A biosynthesis [ 18 ]. Combined with these data, the unidentified ligand bound to E. coli UspE is probably related to an intermediate in lipid A biosynthesis, similar to UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylases.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure and functional implications of the tandem-type universal stress protein UspE from Escherichia coli

Guo

Jin

et al. 2016

BMC Struct Biol

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BackgroundThe universal stress proteins (USP) family member UspE is a tandem-type USP that consists of two Usp domains. The UspE expression levels of the Escherichia coli (E. coli) become elevated in response to oxidative stress and DNA damaging agents, including exposure to mitomycin C, cadmium, and hydrogen peroxide. It has been shown that UspA family members are survival factors during cellular growth arrest. The structures and functions of the UspA family members control the growth of E. coli in animal hosts. While several UspA family members have known structures, the structure of E. coli UspE remains to be elucidated.ResultsTo understand the biochemical function of UspE, we have determined the crystal structure of E. coli UspE at 3.2 Å resolution. The asymmetric unit contains two protomers related by a non-crystallographic symmetry, and each protomer contains two tandem Usp domains. The crystal structure shows that UspE is folded into a fan-shaped structure similar to that of the tandem-type Usp protein PMI1202 from Proteus mirabilis, and it has a hydrophobic cavity that binds its ligand. Structural analysis revealed that E. coli UspE has two metal ion binding sites, and isothermal titration calorimetry suggested the presence of two Cd2+ binding sites with a Kd value of 38.3–242.7 μM. Structural analysis suggested that E. coli UspE has two Cd2+ binding sites (Site I: His117, His 119; Site II: His193, His244).ConclusionThe results show that the UspE structure has a hydrophobic pocket. This pocket is strongly bound to an unidentified ligand. Combined with a previous study, the ligand is probably related to an intermediate in lipid A biosynthesis. Subsequently, sequence analysis found that UspE has an ATP binding motif (Gly269- X2-Gly272-X9-Gly282-Asn) in its C-terminal domain, which was confirmed by in vitro ATPase activity monitored using Kinase-Glo® Luminescent Kinase Assay. However, the residues constituting this motif were disordered in the crystal structure, reflecting their intrinsic flexibility. ITC experiments revealed that the UspE probably has two Cd2+ binding sites. The His117, His 119, His193, and His244 residues within the β-barrel domain are necessary for Cd2+ binding to UspE protein. As mentioned above, USPs are associated with several functions, such as cadmium binding, ATPase function, and involvement in lipid A biosynthesis by some unknown way.

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