MAO and LSD1 Inhibitor Characterization

Rosiglitazone was purchased from Cayman Chemical, pioglitazone and troglitazone from Sigma-Aldrich. MAO A and B recombinant proteins were over-expressed in Pichia pastoris and purified following previously published procedures.^{15 ,16} Enzymatic activities were measured spectrophotometrically using the horseradish peroxidase coupled Amplex red assay (ΔⲈ = 54,000 M⁻¹-cm⁻¹, λ = 560 nm) with p-CF₃-benzylamine and benzylamine or phenethylamine as substrates for MAO A and MAO B, respectively. Inhibitor Ki values were determined by measuring the initial rates of substrate oxidation (six different concentrations) in the presence of varying concentrations of inhibitor (a minimum of four different concentrations). K_i values were determined using global fit analysis of the hyperbolic fits of enzyme activity with inhibitor concentrations using Graphpad Prism 5.0 software. Crystals of MAO B complexes were grown under conditions described previously^{14 (link)} in the presence of ~500 μM inhibitors. X-ray diffraction data were collected at the ESRF (Grenoble, France) and at the SLS (Villigen, Switzerland) synchrotrons. Data processing and structure refinement were performed using programs of the CCP4 package following standard protocols (Table S1).^{25 (link)} Structural representations were generated with CCP4mg.^{26 (link)} Purification of recombinant human LSD1/CoREST complex and inhibition assays against this enzyme were carried out as described.^{27 (link)}