An immuno-chemo-proteomics method for drug target deconvolution.
Chemical proteomics is an emerging technique for drug target deconvolution and profiling the toxicity of known drugs. With the use of this technique, the specificity of a small molecule inhibitor toward its potential targets can be characterized and information thus obtained can be used in optimizing lead compounds. Most commonly, small molecules are immobilized on solid supports and used as affinity chromatography resins to bind targets. However, it is difficult to evaluate the effect of immobilization on the affinity of the compounds to their targets. Here, we describe the development and application of a soluble probe where a small molecule was coupled with a peptide epitope which was used to affinity isolate binding proteins from cell lysate. The soluble probe allowed direct verification that the compound after coupling with peptide epitope retained its binding characteristics. The PKC-alpha inhibitor Bisindolylmaleimide-III was coupled with a peptide containing the FLAG epitope. Following incubation with cellular lysates, the compound and associated proteins were affinity isolated using anti-FLAG antibody beads. Using this approach, we identified the known Bisindolylmaleimide-III targets, PKC-alpha, GSK3-beta, CaMKII, adenosine kinase, CDK2, and quinine reductase type 2, as well as previously unidentified targets PKAC-alpha, prohibitin, VDAC and heme binding proteins. This method was directly compared to the solid-phase method (small molecule was immobilized to a solid support) providing an orthogonal strategy to aid in target deconvolution and help to eliminate false positives originating from nonspecific binding of the proteins to the matrix.
Saxena
Chaitanya
C
Integrative Biology, Greenfield Laboratories, Eli Lilly and Company, Greenfield, IN 46140, USA. saxena_chaitanya@lilly.com
Zhen
Eugene
E
Higgs
Richard E
RE
Hale
John E
JE
D000595:Amino Acid Sequence; D000906:Antibodies; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000737:chemistry; D016503:Drug Delivery Systems; D000939:Epitopes; D004853:Epoxy Resins; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics
D000906:Antibodies; D003432:Cross-Linking Reagents; D000939:Epitopes; D004853:Epoxy Resins; D007211:Indoles; D008301:Maleimides; D009842:Oligopeptides; D010455:Peptides; D011506:Proteins; C531942:bisindolylmaleimide III; C085706:FLAG peptide; D051571:Protein Kinase C-alpha