Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε‐amino group of lysine or hydroxylysine on collagen side‐chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS‐5153A, a novel mechanism based, fast‐acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS‐5153A dose‐dependently reduced LOXL2‐mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet‐induced, PXS‐5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS‐5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.

Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copperdependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, longlasting inhibition of the enzyme after a single low dose in vivo.PXS-4681A irreversibly inhibits the enzyme with an apparent K i of 37 nM and a k inact of 0.26 min 21 with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-a, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.

Background and purposeThe persistent influx of neutrophils into the lung and subsequent tissue damage are characteristics of COPD, cystic fibrosis and acute lung inflammation. VAP-1/SSAO is an endothelial bound adhesion molecule with amine oxidase activity that is reported to be involved in neutrophil egress from the microvasculature during inflammation. This study explored the role of VAP-1/SSAO in neutrophilic lung mediated diseases and examined the therapeutic potential of the selective inhibitor PXS-4728A.MethodsMice treated with PXS-4728A underwent intra-vital microscopy visualization of the cremaster muscle upon CXCL1/KC stimulation. LPS inflammation, Klebsiella pneumoniae infection, cecal ligation and puncture as well as rhinovirus exacerbated asthma models were also assessed using PXS-4728A.ResultsSelective VAP-1/SSAO inhibition by PXS-4728A diminished leukocyte rolling and adherence induced by CXCL1/KC. Inhibition of VAP-1/SSAO also dampened the migration of neutrophils to the lungs in response to LPS, Klebsiella pneumoniae lung infection and CLP induced sepsis; whilst still allowing for normal neutrophil defense function, resulting in increased survival. The functional effects of this inhibition were demonstrated in the RV exacerbated asthma model, with a reduction in cellular infiltrate correlating with a reduction in airways hyperractivity.Conclusions and implicationsThis study demonstrates that the endothelial cell ligand VAP-1/SSAO contributes to the migration of neutrophils during acute lung inflammation, pulmonary infection and airway hyperractivity. These results highlight the potential of inhibiting of VAP-1/SSAO enzymatic function, by PXS-4728A, as a novel therapeutic approach in lung diseases that are characterized by neutrophilic pattern of inflammation.

The consumption of flavonoid-rich foods, in particular fruits and vegetables, has been epidemiologically associated with a reduced risk of heart disease, neurodegenerative disease, cancer and other chronic diseases. Flavonoid glycosides, the main class of flavonoids, have been shown to exert CNS-mediated activities, particularly as sedative-hypnotics, analgesics or both, nevertheless no studies have evaluated these agents in anxiety. This study assessed the potential anxiolytic effect of three flavonoid glycosides, myrcitrin, naringin and gossypin, in the elevated plus maze test (EPM). Myricitrin (1 mg/kg) was effective on the EPM showing a clear anxiolytic effect with no signs of sedation. However, higher doses showed possible sedative and myorelaxation effects. Gossypin and naringin both shared a similar profile, with low doses (1 mg/kg) inducing a robust anxiolytic effect which diminished with increasing doses of the flavonoids. Higher doses of these two flavonoids showed a dramatic increase in the open arm exploration accompanied by a decrease in locomotor activity. Hence, naringin (30 mg/kg) and gossypin (30 mg/kg) induce both anxiolytic and sedative effects. These results suggest that flavonoid glycosides have the potential to exert a range of CNS-mediated biological activities.

BACKGROUNDG protein-coupled inwardly rectifying potassium (KIR3) channels are important proteins that regulate numerous physiological processes including excitatory responses in the CNS and the control of heart rate. Flavonoids have been shown to have significant health benefits and are a diverse source of compounds for identifying agents with novel mechanisms of action. EXPERIMENTAL APPROACHThe flavonoid glycoside, naringin, was evaluated on recombinant human KIR3.1-3.4 and KIR3.1-3.2 expressed in Xenopus oocytes using two-electrode voltage clamp methods. In addition, we evaluated the activity of naringin alone and in the presence of the KIR3 channel blocker tertiapin-Q (0.5 nM, 1 nM and 3 nM) at recombinant KIR3.1-3.4 channels. Site-directed mutagenesis was used to identify amino acids within the M1-M2 loop of the KIR3.1 F137S mutant channel important for naringin's activity. KEY RESULTSNaringin (100 mM) had minimal effect on uninjected oocytes but activated KIR3.1-3.4 and KIR3.1-3.2 channels. The activation by naringin of KIR3.1-3.4 channels was inhibited by tertiapin-Q in a competitive manner. An alanine-scan performed on the KIR3.1 F137S mutant channel, replacing one by one aromatic amino acids within the M1-M2 loop, identified tyrosines 148 and 150 to be significantly contributing to the affinity of naringin as these mutations reduced the activity of naringin by 20-and 40-fold respectively. CONCLUSIONS AND IMPLICATIONSThese results show that naringin is a direct activator of KIR3 channels and that tertiapin-Q shares an overlapping binding site on the KIR3.1-3.4. This is the first example of a ligand that activates KIR3 channels by binding to the extracellular M1-M2 linker of the channel. AbbreviationsCGP36742 or SGS742, 3-aminopropyl-n-butylphosphinic acid; GPCRs, G protein-coupled receptors; KIR3, G proteincoupled inwardly rectifying potassium channels; LPA, lysophosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; r KIR1.1, rat renal outer medullary potassium; TPN-Q, tertiapin-Q

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