Quantitative proteomics approach for identifying protein-drug interactions in complex mixtures using protein stability measurements.
Knowledge about the protein targets of therapeutic agents is critical for understanding drug mode of action. Described here is a mass spectrometry-based proteomics method for identifying the protein target(s) of drug molecules that is potentially applicable to any drug compound. The method, which involves making thermodynamic measurements of protein-folding reactions in complex biological mixtures to detect protein-drug interactions, is demonstrated in an experiment to identify yeast protein targets of the immunosuppressive drug, cyclosporin A (CsA). Two of the ten protein targets identified in this proof of principle work were cyclophilin A and UDP-glucose-4-epimerase, both of which are known to interact with CsA, the former through a direct binding event (K(d) approximately 70 nM) and the latter through an indirect binding event. These two previously known protein targets validate the methodology and its ability to detect both the on- and off-target effects of protein-drug interactions. The other eight protein targets discovered here, which include several proteins involved in glucose metabolism, create a new framework in which to investigate the molecular basis of CsA side effects in humans.
West
Graham M
GM
Department of Chemistry, Duke University, Durham, NC 27708, USA.
Tucker
Chandra L
CL
Xu
Tao
T
Park
Sung Kyu
SK
Han
Xuemei
X
Yates
John R
JR
Fitzgerald
Michael C
MC
D002853:Chromatography, Liquid; D045424:Complex Mixtures / Q000737:chemistry*; D016572:Cyclosporine / Q000378:metabolism; D013058:Mass Spectrometry; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013816:Thermodynamics; D014534:UDPglucose 4-Epimerase / Q000378:metabolism; D015003:Yeasts
D045424:Complex Mixtures; D004364:Pharmaceutical Preparations; D011506:Proteins; D016572:Cyclosporine; D014534:UDPglucose 4-Epimerase