Target identification using drug affinity responsive target stability (DARTS)

Abstract

Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule-protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein-metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.

Fig. 1.

The DARTS method for drug target identification. (A) Scheme of DARTS. (B) Proof of principle. Recombinant human FKBP12 was incubated with indicated drugs and digested with subtilisin. (C) DARTS with a micromolar mTOR kinase inhibitor (E4). Purple arrow, recombinant human TOR fragments protected from thermolysin proteolysis; *, nonspecific band.

Fig. 2.

DARTS using whole-cell lysate. (A) Intact Jurkat cells were treated with DB (1 μg/mL), and lysates were subjected to thermolysin digestion and Coomassie (SimplyBlue)-staining. (B) Enrichment of EF-1α isoforms in the protected band from A revealed by mass spectrometry analysis (SI Text and Fig. S6). Red, protein enriched >2-fold with P value <0.001; green, protein depleted >2-fold with P value <0.001; blue, unchanged protein. (C) DARTS detection via immunoblotting. GAPDH was resistant to thermolysin under the condition and served as a loading indicator.

Fig. 3.

DARTS identifies a molecular target of resveratrol. (A) Chemical structure of resveratrol. (B) Yeast cell lysates and human HeLa cell lysates were each treated with resveratrol in vitro, followed by thermolysin digestion and silver staining. Protected bands of similar size were detected. (C) Resveratrol protects the wild-type eIF4A, but not the A64Q-substituted eIF4A mutant protein, from proteolysis. (D) Resveratrol inhibits eIF4A-dependent translation in HEK 293 cells as assayed by bicistronic translation reporters. The EMCV IRES requires the eIF4A and eIF4G subunits of eIF4F, whereas the HCV IRES does not (55). (E) eIF4A is required for longevity in resveratrol-treated animals. Resveratrol (50 μM) lengthens the lifespan of wild-type N2 worms fed control (gfp) RNAi (green), but not worms fed eIF4A (inf-1) RNAi (red) or daf-16 RNAi (blue). gfp(RNAi), m_Veh = 19 (n = 74), m_RSV = 20 (n = 78), ***, P = 0.0006; inf-1(RNAi), m_Veh = 26 (n = 76), m_RSV = 24 (n = 79), P = 0.4687; daf-16(RNAi), m_Veh = 17 (n = 78), m_RSV = 17 (n = 76), P = 0.3305. m, mean lifespan (days of adulthood); n, number of animals tested.

Fig. 4.

DARTS using cDNAs. (A) Plasmid cDNA is used to program IVT for DARTS. (B) FKBP12-rapamycin protects translated mTOR fragment in DARTS. Streptavidin-HRP was used to detect biotin-Lys incorporated into the translation product. β-Actin was less susceptible to thermolysin under the condition and served as loading indicator. (C) DARTS with IVT FLAG-tagged mTOR.