Disorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem’s biological activity, the target protein was identified via combined drug affinity responsive target stability and LC–MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload.

Autophagy is a critical regulator of cellular homeostasis, dysregulation of which is associated with diverse diseases. Here we show therapeutic effects of a novel autophagy enhancer identified by high-throughput screening of a chemical library against metabolic syndrome. An autophagy enhancer increases LC3-I to LC3-II conversion without mTOR inhibition. MSL, an autophagy enhancer, activates calcineurin, and induces dephosphorylation/nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy gene expression. MSL accelerates intracellular lipid clearance, which is reversed by lalistat 2 or Tfeb knockout. Its administration improves the metabolic profile of ob/ob mice and ameliorates inflammasome activation. A chemically modified MSL with increased microsomal stability improves the glucose profile not only of ob/ob mice but also of mice with diet-induced obesity. Our data indicate that our novel autophagy enhancer could be a new drug candidate for diabetes or metabolic syndrome with lipid overload.

The drug FK506 (tacrolimus, fujimycin) exerts its immunosuppressive effects by regulating the nuclear factor of the activated T-cell (NFAT) family of transcription factors. However, FK506 also exhibits neuroprotective effects, but its direct target proteins that mediate these effects have not been determined. To identify the target proteins responsible for FK506's neuroprotective effects, the drug affinity responsive target stability (DARTS) method was performed using label-free FK506, and LC-MS/MS analysis of the FK506-treated proteome was also performed. Using DARTS and LC-MS/MS analyses in combination with reference studies, V-ATPase catalytic subunit A (ATP6V1A) was identified as a new target protein of FK506. The biological relevance of ATP6V1A in mediating the neuroprotective effects of FK506 was validated by analyzing FK506 activity with respect to autophagy via acridine orange staining and transcription factor EB (TFEB) translocation assay. These analyses demonstrated that the binding of FK506 with ATP6V1A induces autophagy by activating the translocation of TFEB from the cytosol into the nucleus. Because autophagy has been identified as a mechanism for treating neurodegenerative diseases and because we have demonstrated that FK506 induces autophagy, this study demonstrates that FK506 is a possible new therapy for treating neurodegenerative diseases.

Macroautophagy/autophagy (hereafter autophagy), the process of mass degradation of unnecessary elements within the cell, is often dysregulated in many diseases such as cancer, atherosclerosis, and neurodegenerative diseases. Hence, autophagy modulating agents have a great potential to be therapeutic agents for the autophagy-related diseases. Here we report that an anti-depressant drug sertraline (Sert) is an autophagy-inducing agent. Mechanistically, Sert potentially binds to and antagonizes the mitochondrial VDAC1 (voltage dependent anion channel 1), resulting in reduced cellular ATP (adenosine triphosphate) level, activation of AMP-activated protein kinase (AMPK) and inhibition of its downstream, MTOR (mechanistic target of rapamycin kinase)-RPS6KB1 (ribosomal protein S6 kinase B1) signaling pathway. Cells lacking VDAC1 expression completely abrogate the modulatory effect of Sert on AMPK-MTOR pathway and autophagy-inducing activity. We further show that Sert suppresses tauopathy by promoting the autophagic degradation of MAPT (microtubule associated protein tau) protein via inducing autophagy. Our study demonstrates the potential of Sert as a novel small molecule autophagy-inducing agent and provides a new drug candidate to treat autophagy related diseases by targeting VDAC1. Abbreviations: AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; Baf: bafilomycin A 1 ; BiFC: biomolecular fluorescence complementation; CAMKK2/CAMKKB: calcium/calmodulin dependent protein kinase kinase 2; CC: compound C; DARTS: drug affinity responsive target stability; HUVECs: human umbilical vein endothelial cells; Inda: indatraline; STK11/LKB1: serine/threonine kinase 11; MAPT: microtubule associated protein tau; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; 3-MA: 3-methyladenine; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; Rapa: rapamycin; Sert: sertraline; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; SLC6A4/SERT1: solute carrier family 6 member 4; TFEB: transcription factor EB; VDAC1: voltage dependent anion channel 1; WT: wild-type; WM: wortmannin.

Identifying the target proteins of bioactive small molecules is a key step in understanding mode-of-action of the drug and addressing the underlying mechanisms responsible for a particular phenotype. Proteomics has been successfully used to elucidate the target protein profiles of unmodified and ligand-modified bioactive small molecules. In the latter approach, compounds can be modified via click chemistry and combined with activity-based protein profiling. Target proteins are then enriched by performing a pull-down with the modified ligand. Methods that utilize unmodified bioactive small molecules include the cellular thermal shift assay, thermal proteome profiling, stability of proteins from rates of oxidation, and the drug affinity responsive target stability (DARTS) determination (or read-out). This review highlights recent proteomic approaches utilizing data-dependent analysis and data-independent analysis to identify target proteins by DARTS. When combined with liquid chromatography/tandem mass spectrometry, DARTS enables the identification of proteins that bind to drug molecules that leads to a conformational change in the target protein(s). In addition, an effective strategy is proposed for selecting the target protein(s) from within the pool of analyzed candidates. With additional complementary methods, the biologically relevant target proteins that bind to the small bio-active molecules can be further validated.