The lysyl oxidase like 2/3 enzymatic inhibitor, PXS-5153A, reduces crosslinks and ameliorates fibrosis.

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.

Effects of an anti-inflammatory VAP-1/SSAO inhibitor, PXS-4728A, on pulmonary neutrophil migration.

BACKGROUND AND PURPOSE: The 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. METHODS: Mice 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. RESULTS: Selective 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 IMPLICATIONS: This 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.

PXS-4681A, a potent and selective mechanism-based inhibitor of SSAO/VAP-1 with anti-inflammatory effects in vivo.

Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copper-dependent 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, long-lasting inhibition of the enzyme after a single low dose in vivo. PXS-4681A irreversibly inhibits the enzyme with an apparent Ki of 37 nM and a kinact of 0.26 min(-1) 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-α, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.

Hesperidin induces antinociceptive effect in mice and its aglycone, hesperetin, binds to µ-opioid receptor and inhibits GIRK1/2 currents.

This paper extended the evaluation of the depressant and antinociceptive activities of hesperidin in order to determine its effectiveness by the intraperitoneal and oral routes, its pharmacological interaction with diverse pathways of neurotransmission and the role of its aglycone, hesperetin. The capacity of hesperidin and hesperetin to bind to µ-opioid receptor and their actions on µ-opioid receptor co-expressed with GIRK1/GIRK2 channels (G protein-activated inwardly rectifying K+ channels) in Xenopus laevis oocytes were also determined. Hesperidin exhibited a depressant activity in the hole board and locomotor activity tests, antinociceptive activities in the abdominal writhing and hot plate tests and no motor incoordination in the inverted screen and rotarod assays, only by the intraperitoneal route. Hesperetin did not show any effects in vivo in mice in these models, but in vitro it displaced the [³H]DAMGO binding with low-affinity and inhibited inward currents through the expressed GIRK1/2 channels. Although hesperidin actions in vivo demonstrated to be mediated by an opioid mechanism of action, it failed to directly bind to and activate the µ-opioid receptor or produce any change on inward GIRK1/2 currents in vitro. However, it should be considered that hesperidin may be metabolized, possibly resulting in crucial changes in its biological activity.

Naringin directly activates inwardly rectifying potassium channels at an overlapping binding site to tertiapin-Q.

BACKGROUND: G protein-coupled inwardly rectifying potassium (K(IR) 3) 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 APPROACH: The flavonoid glycoside, naringin, was evaluated on recombinant human K(IR) 3.1-3.4 and K(IR) 3.1-3.2 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 K(IR) 3 channel blocker tertiapin-Q (0.5 nM, 1 nM and 3 nM) at recombinant K(IR) 3.1-3.4 channels. Site-directed mutagenesis was used to identify amino acids within the M1-M2 loop of the K(IR) 3.1(F137S) mutant channel important for naringin's activity. KEY RESULTS: Naringin (100 µM) had minimal effect on uninjected oocytes but activated K(IR) 3.1-3.4 and K(IR) 3.1-3.2 channels. The activation by naringin of K(IR) 3.1-3.4 channels was inhibited by tertiapin-Q in a competitive manner. An alanine-scan performed on the K(IR) 3.1(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 IMPLICATIONS: These results show that naringin is a direct activator of K(IR) 3 channels and that tertiapin-Q shares an overlapping binding site on the K(IR) 3.1-3.4. This is the first example of a ligand that activates K(IR) 3 channels by binding to the extracellular M1-M2 linker of the channel.