In Vitro Enzymatic Assay (Sirtuin Biochemical Deacetylation): Sirtuin Biochemical Deacetylation Assays. Sirtuin activity was assessed using the Fleur de Lys assay with recombinant active enzymes SIRT1 (BioMol-SE-239), SIRT2 (BioMol-SE-251),  and the catalytically active fragment of SIRT3 (BioMol-SE-251), as described previously.7,11 Results were measured using a Perkin-Elmer Victor2V 1420 Multilabel plate reader (excitation 355 nm, emission 460 nm). Assays were performed using the manufacturer’s recommendations, and each compound concentration was tested in triplicate. For each experiment, SIRT1 activity was normalized to 1 U/reaction well and SIRT2 and SIRT3 activity to 5 U/reaction well (where U = 1 pmol/min at 37 C, 250 μM substrate, 500 μM NADth). Each reaction well contained enzyme, 500 μM NADth (BioMol-KI-282), 250 μM fluorogenic deacetylase substrate (BioMol-KI-177), supplied reaction buffer, and the compound of interest or mock control (DMSO) in a total volume of 50 μL. Autofluorescent backgrounds were measured in triplicate in reaction solutions containing substrate, buffer, and NADth in triplicate and subtracted from experimental signals.

In Vitro Cellular Assay: Neuronal nuclear antigen (NeuN)-positive neurons and some astroglia are derived from mechanically dissociated ganglionic eminences of E16 rat embryos. The HD model is based on the expression of mutant huntingtin. Treatments of cultures with AK-7 are at 10 μM for 24 h unless stated otherwise. DMSO is included at the same concentrations as a control. Lower dose, chronic treatments with AK-7 are introduced to neurons at DIV4 and continued weekly coinciding with normal medium change.

In Vivo Animal Experiment (Detection of AK-7 in Mouse Brain): AK-7, solubilized at 1.5 mg/mL in 25% Cremophor EL / 10% DMSO in water, is administered by intraperitoneal injection to 11 week old mice at 15 mg/kg/dose, and compound levels in serum and brain are measured following sacrifice. Blood is collected and centrifuged at 7,000 rpm for 7 min, and then serum is aspirated and immediately frozen in liquid nitrogen. Brains are immediately frozen in liquid nitrogen and stored at –80°C. Brains are weighed and then homogenized in four volumes of 10% Cremophor RH40 in water using a Polytron homogenizer, and 2% v/v phosphoric acid is added to the homogenate, vortexed, and centrifuged at 10,000 g at 25°C for 1 h. The supernatant is aspirated, and solid phase extraction is performed immediately. Serum samples are vortexed into 2% v/v phosphoric acid and centrifuged at 2500 rpm for 10 min. Samples (total collected serum or 1 mL homogenate supernatant) were then spiked with an internal standard of N-(4-bromo-phenyl)-3-(4-bromo-phenylsulfamoyl)-benzamide/DMSO to 5 μM and passed through 1 mL Oasis HLB SPE cartridges (Waters), preconditioned with 1mL of methanol followed by 1mL of water. The adsorbent was washed with three volumes of 5% methanol in water, and the sample was eluted

with 0.5 mL of methanol and stored at
80 C until HPLC.

Reference: A brain-permeable small molecule reduces neuronal cholesterol by inhibiting activity of sirtuin 2 deacetylase. ACS Chem Biol. 2011 Jun 17;6(6):540-6.