AK-7 is a brain-permeable SIRT2 inhibitor and to characterize its cholesterol-reducing properties in neuronal models with an IC50 of 15.5 μM.

SIRT2:15.5 μM

AK-7 is a selective cell- and brain-permeable SIRT2 inhibitor,this SIRT2 inhibitor stimulated cytoplasmic retention of sterol regulatory element binding protein-2 and subsequent transcriptional downregulation of cholesterol biosynthesis genes, resulting in reduced total cholesterol in primary striatal neurons. Furthermore, the identified inhibitor reduced cholesterol in cultured na?ve neuronal cells and brain slices from wild-type mice[1].AK-7 has roles in metabolic diseases, cancer, age-related disorders, and neurodegenerative diseases, potentially including Alzheimer's, Huntington's, and Parkinson's diseases[2][3].

AK-7 (15 mg/kg/dose, i.p.) is brain-permeable in wild-type and HD mice[1].

Sirtuin activity was assessed using the Fleur de Lys assay with recombinant active enzymes SIRT1, SIRT2, and the catalytically active fragment of SIRT3. Results were measured using a 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 NAD+). Each reaction well-contained enzyme, 500 μM NAD+, 250 μM fluorogenic deacetylase substrate, 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 NAD+ in triplicate and subtracted from experimental signals [1].

Neuronal nuclear antigen (NeuN)-positive neurons and some astroglia were derived from mechanically dissociated ganglionic eminences of E16 rat embryos. The HD model based on the expression of mutant huntingtin has been described previously. Treatments of cultures with AK-7 were at 10 μM for 24 h unless stated otherwise. DMSO was included at the same concentrations as a control. Lower dose, chronic treatments with AK-7 were introduced to neurons at DIV4 and continued weekly coinciding with normal medium change [1].

AK-7, solubilized at 1.5 mg/mL in 25% Cremophor EL (BASF)/ 10% DMSO in water, was administered by intraperitoneal injection to 11 week old mice at 15 mg/kg/dose. Blood was collected and centrifuged at 7,000 rpm for 7 min, and then serum was aspirated and immediately frozen in liquid nitrogen. Brains were immediately frozen in liquid nitrogen and stored at -80 C. Brains were weighed and then homogenized in four volumes of 10% Cremophor RH40 in water using homogenizer, and 2% v/v phosphoric acid was added to the homogenate, vortexed, and centrifuged at 10,000g at 25 C for 1 h. The supernatant was aspirated, and solid phase extraction was performed immediately. Serum samples were vortexed into 2% v/v phosphoric acid and centrifuged at 2500 rpm for 10 min[1]