Inhibitor affinity chromatography: profiling the specific reactivity of the proteome with immobilized molecules.
An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor-protein interactions as a complementary approach to two-dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin-dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme. Molecular interactions with the Cdk2 inhibitor were compared for HCT116 cancer cells versus rat pancreatic acinar cells. Proteins interacting with the ligand on the IAC matrix were identified by mass spectrometry. Isothermal calorimetry was used to confirm and quantitatively evaluate the binding affinity of some of the interacting proteins. Heat-shock protein (Hsp) 70 and Hsp27 were the strongest interactors with the inhibitor, displaying binding affinities comparable to those of Cdk2. These results support the use of IAC as a general method for the rapid identification and qualitative evaluation of the in vivo targets and potential side effects of a given drug.
Lolli
Graziano
G
Biology Department, Discovery Research Oncology, Pharmacia Corporation, Viale Pasteur 10, 20014 Nerviano, Milan, Italy.
Thaler
Florian
F
Valsasina
Barbara
B
Roletto
Fulvia
F
Knapp
Stefan
S
Uggeri
Mauro
M
Bachi
Angela
A
Matafora
Vittoria
V
Storici
Paola
P
Stewart
Albert
A
Kalisz
Henryk M
HM
Isacchi
Antonella
A
D000818:Animals; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases / Q000037:antagonists & inhibitors; D002151:Calorimetry; D002460:Cell Line; D045744:Cell Line, Tumor; D002845:Chromatography / Q000379:methods*; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007313:Insecta; D008024:Ligands; D013058:Mass Spectrometry; D008956:Models, Chemical; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D020543:Proteome; D051381:Rats; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014357:Trypsin / Q000494:pharmacology
D019925:Cyclin A; D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; C486114:Hspb1 protein, rat; D008024:Ligands; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D011108:Polymers; D020543:Proteome; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D014357:Trypsin