ABSTRACT
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
Human vascular adhesion protein 1 (VAP-1) is involved in lymphocyte-endothelial cell adhesion and has been implicated in many human inflammatory diseases. VAP-1 is a member of the copper amine oxidase family of enzymes with a trihydroxyphenylalanine quinone (TPQ) cofactor. Previously characterized crystals of VAP-1 suffered from anisotropy and contained disordered regions; in addition, one form was consistently twinned. In an effort to grow crystals that diffracted to higher resolution for inhibitor-binding studies, a construct with an N-terminal deletion was made and expressed in the Chinese hamster ovary (CHO) glycosylation mutant cell line Lec8. Screening produced crystals that displayed some anisotropy and contained seven molecules per asymmetric unit. These crystals belonged to space group C2, with unit-cell parameters a=394.5, b=115.8, c=179.3â Å, ß=112.3°. The structure was refined to a resolution of 2.9â Å, with Rcryst and Rfree values of 0.250 and 0.286, respectively.
Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Cell Adhesion Molecules/chemistry , Animals , CHO Cells , Catalytic Domain , Cricetinae , Cricetulus , Crystallography, X-Ray , Cysteine/chemistry , Disulfides/chemistry , Glycosylation , Humans , Models, Molecular , Protein Binding , Protein MultimerizationABSTRACT
Mechanistic and structural studies have been carried out to investigate the molecular basis for the irreversible inhibition of human MAO-B by mofegiline. Competitive inhibition with substrate shows an apparent K(i) of 28 nM. Irreversible inhibition of MAO-B occurs with a 1:1 molar stoichiometry with no observable catalytic turnover. The absorption spectral properties of mofegiline inhibited MAO-B show features (lambda(max) approximately 450 nm) unlike those of traditional flavin N(5) or C(4a) adducts. Visible and near-UV circular dichroism spectra of the mofegiline-MAO-B adduct shows a negative peak at 340 nm with an intensity similar to that of N(5) flavocyanine adducts. The X-ray crystal structure of the mofegiline-MAO-B adduct shows a covalent bond between the flavin cofactor N(5) with the distal allylamine carbon atom as well as the absence of the fluorine atom. A mechanism to explain these structural and spectral data is proposed.