Discovery of Potent and Selective Inhibitors of Human Reticulocyte 15-Lipoxygenase-1

Rai, Ganesha; Kenyon, Victor; Jadhav, Ajit; Schultz, Lena; Armstrong, Michelle; Jameson, J. Brian; Hoobler, Eric K.; Leister, William; Simeonov, Anton; Holman, Theodore R.; Maloney, David J.

doi:10.1021/jm1008852

Cited by 80 publications

(103 citation statements)

References 61 publications

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“…56 Moreover, this compound possessed an essential ester moiety that could be susceptible to intracellular and plasma esterases, rendering it inactive and possibly limiting its utility in advanced biological models. Thus, we were encouraged by the in vitro ADME (Table 4) and in vivo PK properties (Table 5) of 1 , as they represent a vast improvement over the majority of compounds reported previously (vide infra).…”

Section: Resultsmentioning

confidence: 99%

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Potent and Selective Inhibitors of Human Reticulocyte 12/15-Lipoxygenase as Anti-Stroke Therapies

et al. 2014

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A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound 1 (ML351), a novel chemotype for 12/15-LOX inhibition that has nanomolar potency (IC50 = 200 nM) against human 12/15-LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT22 cells. In addition, it exhibited greater than 250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Lastly, 1 significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept mouse models of stroke.

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Section: Resultsmentioning

confidence: 99%

“…56,79 The inhibitor concentrations of 0, 0.05, 2, and 5 μM were used. Reactions were initiated by adding approximately 40–60 nM 12/15-LOX to a constantly stirring 2 mL cuvette containing 1–20 μM AA in 25 mM HEPES buffer (pH 7.5) in the presence of 0.01% Triton X-100.…”

Section: Methodsmentioning

confidence: 99%

Potent and Selective Inhibitors of Human Reticulocyte 12/15-Lipoxygenase as Anti-Stroke Therapies

et al. 2014

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“…A HTS of approximately 74,000 compounds was performed with h12/15-LOX, as previously reported, 21 and the top 1000 compounds were manually screened using the UV-Vis assay with h12/15-LOX. All molecules were screened at 20 µM inhibitor concentration, with 99089 inhibiting h12/15-LOX greater than 90% at this concentration.…”

Section: 3 Results and Discussionmentioning

confidence: 99%

“…ML094 has low nanomolar potency against recombinant h12/15-LOX in vitro (IC 50 = < 10 nM) but lacks activity in cell-based assays, possibly due to the hydrolyzable nature of the ester moiety. 21 In comparison, ML351 has sub-micromolar potency (IC 50 = 200 nM) and exhibits in vivo activity in mice. 13 In the current work, we expand on our initial h12/15-LOX screen and present another unique chemical scaffold for h12/15-LOX inhibition.…”

Section: 1 Introductionmentioning

confidence: 99%

A potent and selective inhibitor targeting human and murine 12/15-LOX

Armstrong

Freedman

Jung

et al. 2016

Bioorganic & Medicinal Chemistry

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Human reticulocyte 12/15-Lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC50 potency of 3.4 ± 0.5 µM against h12/15-LOX in vitro and an ex vivo IC50 potency of approximately 10 µM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 µM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a Kic of 1.0 ± 0.08 µM and a Kiu of 6.0 ± 3.3 µM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.

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“…The groups at ParkeDavis/Warner-Lambert (now Pfizer) [16][17][18][19][20][21][22] and Bristol-Myers Squibb 23-26 used the rabbit reticulocyte 15-LOX-1 for their primary screen, followed by a cell-based assay with recombinant 15-LOX-1 or a rabbit in vivo model to design compounds for the treatment of inflammation and/or atherosclerosis. More recently, Maloney and Holman used 15-LOX-1 in their primary screen to develop molecular tools 27 and compounds aimed for anti-stroke therapy. 8 A selection of 15-LOX-1 inhibitors reported by the groups mentioned above are depicted in Figure 1.…”

Section: Introductionmentioning

confidence: 99%