ML346 is a novel activator of Hsp70.

ML346 has good chemical stability, significantly high water solubility, is not reactive with excess glutathione, and is cell permeable. The probe ML346 will help elucidate roles for activation of Hsp70 and HSF-1 in the prevention and progression of cancers, cellular aging, and metabolic and neurodegenerative disorders. ML346 is a novel modulator of proteostasis for protein conformational diseases.

ML346 activates transcription of the Hsp70 promoter and suppresses aggregation of poly-glutamines in a C. elegans model, suggesting the probe has efficacy in modifying protein aggregation and associated toxicity. ML346 induces HSF-1-dependent chaperone expression and restores protein folding in conformational disease models.

Assays (25.5 μL volume) are carried out robotically at room temperature (21°C) and are linear with respect to time and enzyme concentration under the conditions used. Assays are performed for 30 min using Multidrop Micro reagent dispensers in a 96-well format. The concentration of magnesium acetate in the assays is 10 mM and [γ-33P]ATP (800 c.p.m./pmol) is used at 5, 20 or 50 μM as indicated, in order to be at or below the Kmfor ATP for each enzyme.The assays are initiated with MgATP, stopped by the addition of 5 μL of 0.5 M orthophosphoric acid and spotted on to P81 filter plates using a unifilter harvester. The IC50 values of inhibitors are determined after carrying out assays at ten different concentrations of each compound[2].