Target identification remains challenging for the field of chemical biology. We describe an integrative chemical genomic and proteomic approach combining the use of differentially active analogs of small molecule probes with stable isotope labeling by amino acids in cell culture-mediated affinity enrichment, followed by subsequent testing of candidate targets using RNA interference-mediated gene silencing. We applied this approach to characterizing the natural product K252a and its ability to potentiate neuregulin-1 (Nrg1)/ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4)-dependent neurotrophic factor signaling and neuritogenesis. We show that AAK1 (adaptor-associated kinase 1) is a relevant target of K252a, and that the loss of AAK1 alters ErbB4 trafficking and expression levels, providing evidence for a previously unrecognized role for AAK1 in Nrg1-mediated neurotrophic factor signaling. Similar strategies should lead to the discovery of novel targets for therapeutic development.
Bibliographical noteFunding Information:
We thank members of the Broad Chemical Biology and Proteomics Platforms, as well as the Stanley Center for Psychiatric Research for support, and access to key instrumentation and reagents. This work was supported by grants from the Stanley Medical Research Institute, the Broad Institute SPARC, and the US National Institutes of Health (NIH) (1R21MH087896-01). The project has also been funded in part with funds from the US National Cancer Institute's Initiative for Chemical Genetics (contract number N01-CO-12400) and an NIH grant for Genomics Based Drug Discovery-Target ID project (RL1HG004671), administratively linked to RL1CA133834, RL1GM084437, and UL1RR024924. L.K, S.-E.O., C.J.L., and J.M.M. developed biochemical methods, designed, experiments, and analyzed data. J.L.W. provided advice on chemical inhibitors and synthesized analogs of K252a. J.R.D., T.A.L., and X.W. designed, synthesized, and characterized the K252a-based affinity probes. S.-E.O., L.K., and S.A.C. designed, performed, and interpreted the SILAC-target identification studies. S.D.C. provided key reagents and advice for the AAK1 kinase assay, biochemical studies, and data interpretation. D.A.P. designed and performed the K252a-AAK1 computational studies. S.L.S., S.A.C., E.M.S., and S.J.H. provided funding, mentoring, and advised in experimental design and data interpretation. L.K., J.M.M., and S.J.H. prepared the manuscript, which was edited by all authors. The corresponding author (S.J.H.) certifies that all authors have agreed to all the content in the manuscript, including the data as presented. There were no competing financial interests.