Elucidation of the molecular mechanism and the efficacy in vivo of a novel 1,4‐benzoquinone that inhibits 5‐lipoxygenase

Abstract

BACKGROUND AND PURPOSE1,4-Benzoquinones are well-known inhibitors of 5-lipoxygenase (5-LOX, the key enzyme in leukotriene biosynthesis), but the molecular mechanisms of 5-LOX inhibition are not completely understood. Here we investigated the molecular mode of action and the pharmacological profile of the novel 1,4-benzoquinone derivative 3-((decahydronaphthalen-6-yl)methyl)-2,5-dihydroxycyclohexa-2,5-diene-1,4-dione (RF-Id) in vitro and its effectiveness in vivo. EXPERIMENTAL APPROACHMechanistic investigations in cell-free assays using 5-LOX and other enzymes associated with eicosanoid biosynthesis were conducted, along with cell-based studies in human leukocytes and whole blood. Molecular docking of RF-Id into the 5-LOX structure was performed to illustrate molecular interference with 5-LOX. The effectiveness of RF-Id in vivo was also evaluated in two murine models of inflammation. KEY RESULTSRF-Id consistently suppressed 5-LOX product synthesis in human leukocytes and human whole blood. RF-Id also blocked COX-2 activity but did not significantly inhibit COX-1, microsomal PGE2 synthase-1, cytosolic PLA2 or 12-and 15-LOX. Although RF-Id lacked radical scavenging activity, reducing conditions facilitated its inhibitory effect on 5-LOX whereas cell stress impaired its efficacy. The reduced hydroquinone form of RF-Id (RED-RF-Id) was a more potent inhibitor of 5-LOX as it had more bidirectional hydrogen bonds within the 5-LOX substrate binding site. Finally, RF-Id had marked anti-inflammatory effects in mice in vivo. CONCLUSIONS AND IMPLICATIONSRF-Id represents a novel anti-inflammatory 1,4-benzoquinone that potently suppresses LT biosynthesis by direct inhibition of 5-LOX with effectiveness in vivo. Mechanistically, RF-Id inhibits 5-LOX in a non-redox manner by forming discrete molecular interactions within the active site of 5-LOX.

Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase

“…The same pattern of interference with 5-LOX product formation was observed for the FLAP inhibitor MK886 (IC 50 monocytes: ~3 nM, neutrophils: 10–14 nM; IC 50 5-LOX in cell-free assays: >10 µM, not shown), which is in agreement with the literature 24 . In contrast, the direct 5-LOX inhibitor zileuton inhibited 5-LOX activity in the cell-based (monocytes, neutrophils) and cell-free assays about equally well (IC 50 = 1.5 and 0.8 µM, respectively), as reported 25 . A typical feature of FLAP inhibitors is their loss of efficiency, when cells are stimulated for 5-LOX product formation in the presence of exogenous AA, since (I) FLAP inhibitors compete with AA binding within the active site of FLAP 26 , and (II) ample AA supply may circumvent the requirement of FLAP for cellular 5-LOX product formation 27 , 28 .…”

Finding New Molecular Targets of Familiar Natural Products Using In Silico Target Prediction

“…As positive controls, compound 2–9 (CAS: 745028-76-6) [ 35 ] was used for AKR1C3 assays, compound 24 (CAS: 873206-61-2) [ 34 ] for 17β HSD3 assays, and compound 19 (CAS: 1340482-23-6) [ 72 ] for 17β HSD2 assays, all in 1-µM concentrations. Inhibitory activities towards 5-LO and COX-1 were determined as described earlier by Schaible et al [ 73 ]. and Koeberle et al [ 74 ], respectively.…”

Formulation and Characterization of Solid Lipid Nanoparticles Loading RF22-c, a Potent and Selective 5-LO Inhibitor, in a Monocrotaline-Induced Model of Pulmonary Hypertension

“…Within the context of our investigations toward the synthesis of different compound libraries with prospects for therapeutic use, we recently studied the natural and synthetic quinone derivatives. Over the years, our group has long investigated about the 1,4-benzoquinones class as lead compounds having potent antioxidant, anti-inflammatory, and anticancer properties and highlighted their efficient inhibitory activity against 5-LO (Filosa et al, 2013;Petronzi et al, 2013;Schaible et al, 2014;Filosa et al, 2015;Zappavigna et al, 2016).…”