ABSTRACT
The discovery of a dual MAO-B/SSAO inhibitor PXS-5131 is reported. The compound offers a compact and rigid three-dimensional structure with superior selectivity over MAO-A. Potency and selectivity are linked to both the double bond geometry and stereochemistry of the allylamine moiety, highlighting the importance of optimal set up of these features in the class of amine oxidase inhibitors. PXS-5131 possesses an attractive preclinical pharmacokinetic profile and has anti-inflammatory properties in models of acute inflammation and neuroinflammation.
Subject(s)
Amine Oxidase (Copper-Containing) , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Monoamine Oxidase , Monoamine Oxidase Inhibitors/pharmacologyABSTRACT
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.
Subject(s)
Amino Acid Oxidoreductases/antagonists & inhibitors , Collagen/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Fibrosis/prevention & control , Myocardial Infarction/prevention & control , Non-alcoholic Fatty Liver Disease/prevention & control , Animals , Carbon Tetrachloride/toxicity , Collagen/drug effects , Collagen/metabolism , Cross-Linking Reagents/chemistry , Elastin/antagonists & inhibitors , Elastin/drug effects , Elastin/metabolism , Extracellular Matrix/drug effects , Fibrosis/chemically induced , Fibrosis/enzymology , Fibrosis/pathology , Male , Mice , Mice, Inbred C57BL , Myocardial Infarction/enzymology , Myocardial Infarction/pathology , Non-alcoholic Fatty Liver Disease/enzymology , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/pathology , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Allyl Compounds/pharmacology , Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Anti-Inflammatory Agents/pharmacology , Cell Adhesion Molecules/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Sulfonamides/pharmacology , Adipose Tissue/drug effects , Adipose Tissue/enzymology , Allyl Compounds/chemistry , Allyl Compounds/pharmacokinetics , Allyl Compounds/therapeutic use , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/therapeutic use , Dermatitis/drug therapy , Dermatitis/enzymology , Dermatitis/immunology , Disease Models, Animal , Dogs , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Humans , In Vitro Techniques , Mice , Microsomes/drug effects , Microsomes/enzymology , Models, Biological , Molecular Structure , Pneumonia/drug therapy , Pneumonia/enzymology , Pneumonia/immunology , Rabbits , Rats , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacokinetics , Small Molecule Libraries/therapeutic use , Species Specificity , Sulfonamides/chemistry , Sulfonamides/pharmacokinetics , Sulfonamides/therapeutic useABSTRACT
A new class of 3-fluoroallyl amine-based SSAO/VAP-1 inhibitors is reported. These compounds have excellent selectivity over diamine oxidase, MAO-A and MAO-B. Synthesis and SAR studies leading to compound 28 (PXS-4159A) are reported. The pharmacokinetic profile of 28 in the rat, together with activity in a murine model of lung inflammation are also disclosed.
Subject(s)
Allyl Compounds/chemical synthesis , Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Amines/chemical synthesis , Anti-Inflammatory Agents/chemical synthesis , Monoamine Oxidase Inhibitors/chemical synthesis , Pneumonia/drug therapy , Allyl Compounds/pharmacokinetics , Allyl Compounds/pharmacology , Amine Oxidase (Copper-Containing)/metabolism , Amines/pharmacokinetics , Amines/pharmacology , Animals , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/pharmacology , Drug Discovery , Halogenation , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Mice , Models, Molecular , Monoamine Oxidase Inhibitors/pharmacokinetics , Monoamine Oxidase Inhibitors/pharmacology , Pneumonia/enzymology , Rats , Structure-Activity RelationshipABSTRACT
Lysyl oxidase-like 2 (LOXL2) is a secreted enzyme that catalyzes the formation of cross-links in extracellular matrix proteins, namely, collagen and elastin, and is indicated in fibrotic diseases. Herein, we report the identification and subsequent optimization of a series of indole-based fluoroallylamine inhibitors of LOXL2. The result of this medicinal chemistry campaign is PXS-5120A (12k), a potent, irreversible inhibitor that is >300-fold selective for LOXL2 over LOX. PXS-5120A also shows potent inhibition of LOXL3, an emerging therapeutic target for lung fibrosis. Key to the development of this compound was the utilization of a compound oxidation assay. PXS-5120A was optimized to show negligible substrate activity in vitro for related amine oxidase family members, leading to metabolic stability. PXS-5120A, in a pro-drug form (PXS-5129A, 12o), displayed anti-fibrotic activity in models of liver and lung fibrosis, thus confirming LOXL2 as an important target in diseases where collagen cross-linking is implicated.
Subject(s)
Amines/chemistry , Amines/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Protein-Lysine 6-Oxidase/antagonists & inhibitors , Animals , Drug Design , Humans , Mice , Triazoles/chemistryABSTRACT
Lysyl Oxidase-like 2 (LOXL2), a member of the lysyl oxidase family of amine oxidases is known to be important in normal tissue development and homeostasis, as well as the onset and progression of solid tumors. Here we tested the anti-tumor properties of two generations of novel small molecule LOXL2 inhibitor in the MDA-MB-231 human model of breast cancer. We confirmed a functional role for LOXL2 activity in the progression of primary breast cancer. Inhibition of LOXL2 activity inhibited the growth of primary tumors and reduced primary tumor angiogenesis. Dual inhibition of LOXL2 and LOX showed a greater effect and also led to a lower overall metastatic burden in the lung and liver. Our data provides the first evidence to support a role for LOXL2 specific small molecule inhibitors as a potential therapy in breast cancer.
Subject(s)
Amino Acid Oxidoreductases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Enzyme Inhibitors/pharmacology , Amino Acid Oxidoreductases/genetics , Aminopropionitrile/pharmacology , Animals , Apoptosis , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cancer-Associated Fibroblasts/drug effects , Cancer-Associated Fibroblasts/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Gene Knockdown Techniques , Gene Silencing , Humans , Mice , Neoplasm Metastasis , Neovascularization, Pathologic , Xenograft Model Antitumor AssaysSubject(s)
Amino Acid Oxidoreductases/pharmacology , Antifibrotic Agents/pharmacology , Biosensing Techniques/instrumentation , Biosensing Techniques/standards , Amino Acid Oxidoreductases/analysis , Amino Acid Oxidoreductases/therapeutic use , Antifibrotic Agents/analysis , Antifibrotic Agents/therapeutic use , Biosensing Techniques/methods , Fibrosis/drug therapy , Fibrosis/prevention & control , HumansABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a progressive decline in lung function which can be attributed to excessive scarring, inflammation and airway remodelling. Mannose-6-phosphate (M6P) is a strong inhibitor of fibrosis and its administration has been associated with beneficial effects in tendon repair surgery as well as nerve repair after injury. Given this promising therapeutic approach we developed an improved analogue of M6P, namely PXS64, and explored its anti-fibrotic effects in vitro. Normal human lung fibroblasts (NHLF) and human lung fibroblast 19 cells (HF19) were exposed to active recombinant human TGF-ß1 to induce increases in fibrotic markers. rhTGF-ß1 increased constitutive protein levels of fibronectin and collagen in the NHLF cells, whereas HF19 cells showed increased levels of fibronectin, collagen as well as αSMA (alpha smooth muscle actin). PXS64 demonstrated a robust inhibitory effect on all proteins analysed. IPF patient fibroblasts treated with PXS64 presented an improved phenotype in terms of their morphological appearance, as well as a decrease in fibrotic markers (collagen, CTGF, TGF-ß3, tenascin C, αSMA and THBS1). To explore the cell signalling pathways involved in the anti-fibrotic effects of PXS64, proteomics analysis with iTRAQ labelling was performed and the data demonstrated a specific antagonistic effect on the TGF-ß1 pathway. This study shows that PXS64 effectively inhibits the production of extracellular matrix, as well as myofibroblast differentiation during fibrosis. These results suggest that PXS64 influences tissue remodelling by inhibiting TGF-ß1 signalling in NHLF and HF19 cell lines, as well as in IPF patient fibroblasts. Thus PXS64 is a potential candidate for preclinical application in pulmonary fibrosis.