In vitro activity: SMER28 is a small molecule activator (enhancer/modulator) of autophagy that acts via an mTOR-independent mechanism. It induces autophagy independent of rapamycin in mammalian cells and prevents the accumulation of amyloid beta peptide. The hallmarks of Alzheimer's disease are the aggregates of amyloid-β (Αβ) peptide and tau protein. Autophagy is one major cellular pathway leading to the removal of aggregated proteins. Induction of autophagy by small-molecule enhancers such as SMER28 greatly decreased the levels of Aβ peptide (apparent EC50 of ~10 μM) and APP-CTF (apparent EC50 of ~20 μM) in a γ-secretase-independent manner. Pharmacological inhibition of autophagy led to a significant accumulation of Aβ peptide and APP-CTF and diminished the effect of SMER28. Therefore, SMER28 may have therapeutic potential to be used for the treatment of Alzheimer's disease and other proteinopathies.

Kinase Assay: Of 50,729 compounds tested in a high-throughput screen, a number of small molecule inhibitors and enhancers (SMERs) of the cytostatic effects of rapamycin were identified and subsequently analyzed in a secondary screen in mammalian cells by analyzing the clearance of A53T mutant α-synuclein (a good autophagy substrate) in the absence of rapamycin as putative modulators of autophagy. We confirmed that SMER10, SMER18, and SMER28 were positive regulators of autophagy acting independently of rapamycin. These SMERs increased autophagosome synthesis and enhanced the clearance of model autophagy substrates such as A53T α-synuclein and mutant huntingtin fragments. Autophagy induced by SMERs was mTOR-independent. Furthermore, these SMERs were protective in a Drosophila model of HD. Further screening of the structural analogs of these three SMERs identified 18 additional small molecules that enhanced the clearance of aggregate-prone protein.

Cell Assay: Cells were grown in 4-well slide chambers (Lab Tek; Nalge Nunc, Rochester, NY, USA) and fixed with 4% paraformaldehyde. Cells were then permeabilized in 0.1% Triton X-100 and stained with primary antibodies, followed by FITC-conjugated secondary antibodies. The coverslips were mounted by Prolong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA). The images were aquired using a confocal microscope and the LSM5 3.2 software.