Inhibiting an Epoxide Hydrolase Virulence Factor from <i>Pseudomonas aeruginosa</i> Protects CFTR

Bahl, Christopher D.; Hvorecny, Kelli L.; Bomberger, Jennifer M.; Hammock, Bruce D.; Morisseau, Christophe; Madden, Dean R.

doi:10.1002/ange.201503983

Cited by 3 publications

(5 citation statements)

References 31 publications

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“…An immunoassay selective for the diol product confirmed conversion of 14,15-EET to 14,15-DHET by Cif, but not by the structurally conserved mutant Cif-D129S (Fig. S1B), which is enzymatically inactive (16).…”

Section: Resultsmentioning

confidence: 98%

“…In particular, the administration of a LXA 4 analog in mice challenged intratracheally with P. aeruginosa led to reduced neutrophil infiltration, weight loss, and bacterial burden, resulting in overall lessening of disease severity (15). An alternative strategy may be to inhibit Cif activity with a targeted small-molecule approach (16,37). In addition to blocking the enzymatic degradation of lipid mediators, inhibitors would also block the ability of Cif to subvert rescue of CFTR by recently approved clinical correctors (38).…”

Section: Discussionmentioning

confidence: 99%

“…Wild-type Cif protein (Cif-WT) and Cif-D129S and Cif-E153Q mutants were expressed as described previously (16,17,29). Hydrolysis of the epoxyeicosatrienoic acids by Cif was measured using a modified adrenochrome assay (36,39).…”

Section: Methodsmentioning

confidence: 99%

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Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators

Flitter

Hvorecny

Ono

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A4 (15-epi LXA4), plays a critical role in limiting neutrophil activation and tissue inflammation, thus promoting the return to tissue homeostasis. Here, we show that a secreted P. aeruginosa epoxide hydrolase, cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), can disrupt 15-epi LXA4 transcellular biosynthesis and function. In the airway, 15-epi LXA4 production is stimulated by the epithelial-derived eicosanoid 14,15-epoxyeicosatrienoic acid (14,15-EET). Cif sabotages the production of 15-epi LXA4 by rapidly hydrolyzing 14,15-EET into its cognate diol, eliminating a proresolving signal that potently suppresses IL-8–driven neutrophil transepithelial migration in vitro. Retrospective analyses of samples from patients with CF supported the translational relevance of these preclinical findings. Elevated levels of Cif in bronchoalveolar lavage fluid were correlated with lower levels of 15-epi LXA4, increased IL-8 concentrations, and impaired lung function. Together, these findings provide structural, biochemical, and immunological evidence that the bacterial epoxide hydrolase Cif disrupts resolution pathways during bacterial lung infections. The data also suggest that Cif contributes to sustained pulmonary inflammation and associated loss of lung function in patients with CF.

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Section: Resultsmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators

Flitter

Hvorecny

Ono

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Cif has been shown to decrease the concentration of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) on the apical surface of lung epithelial cells, sabotage the inflammation resolution signaling between the epithelial cells and the immune system, and hinder mucociliary clearance of bacteria [3,4,5]. The crystal structure of Cif showed that it utilizes a His-Tyr pair instead of the canonical Tyr-Tyr pair for epoxide ring orientation and hydrolysis intermediate stabilization [6]. aCif from Acinetobacter nosocomialis was subsequently shown to belong to this new class of Cif-like (Cfl) epoxide hydrolases [7].…”

Section: Introductionmentioning

confidence: 99%

Biochemical and Structural Characterization of Two Cif-Like Epoxide Hydrolases fromBurkholderia cenocepacia

Taher

Hvorecny

Burke

et al. 2021

Preprint

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Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from Pseudomonas aeruginosa. Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homologue aCif from Acinetobacter nosocomialis have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two Cif-like epoxide hydrolases from Burkholderia cenocepacia. Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the α/β hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity.

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“…One of them triggers degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). 9 The CFTR inhibitory factor (Cif), which has putative orthologs in several opportunistic airway pathogens, 10 is an epoxide hydrolase. Its catalytic activity affects host mucociliary and antiviral defenses and appears to facilitate infection of the lungs.…”

Section: Introductionmentioning

confidence: 99%

Nanobody-based binding assay for the discovery of potent inhibitors of CFTR inhibitory factor (Cif)

Vasylieva

Kitamura

Jiang

et al. 2019

Analytica Chimica Acta

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Lead identification and optimization are essential steps in the development of a new drug. It requires cost-effective, selective and sensitive chemical tools. Here, we report a novel method using nanobodies that allows the efficient screening for potent ligands. The method is illustrated with the cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), a virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. 18 nanobodies selective to Cif were isolated by bio-panning from nanobody-phage library constructed from immunized llama. 8 out of 18 nanobodies were identified as potent inhibitors of Cif enzymatic activity with IC 50 s in the range of 0.3-6.4 μM. A nanobody VHH219 showed high affinity (K D =0.08nM) to Cif and the highest inhibitory potency, IC 50 =0.3 μM. A displacement sandwich ELISA (dsELISA) with VHH219 was then developed for classification of synthetic small molecule inhibitors according their inhibitory potency. The developed assay allowed identification of new inhibitor with highest potency reported so far (0.16±0.02 μM). The results from dsELISA assay correlates strongly with a conventional fluorogenic assay (R= 0.9998) in predicting the inhibitory potency of the tested compounds. However, the novel dsELISA is an order of magnitude more sensitive and allows the identification and ranking of potent inhibitors missed by the classic fluorogenic assay method. These data were supported with Octet biolayer interferometry measurements. The novel method described herein relies solely on the binding properties of the specific neutralizing

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Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR

Cited by 3 publications

References 31 publications

Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators

Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators

Biochemical and Structural Characterization of Two Cif-Like Epoxide Hydrolases fromBurkholderia cenocepacia

Nanobody-based binding assay for the discovery of potent inhibitors of CFTR inhibitory factor (Cif)

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