A quantitative affinity-based technique for the identification of potential lead compounds.
Here, we describe the development and validation of a quantitative analytical method for rapid evaluation of protein-compound interactions. The method uses size-exclusion chromatography in a 96-well format with liquid chromatography/mass spectrometry (qSEC-LC/MS) by which the amount of a compound that was originally in complex with a target protein is determined as an indicator of the binding affinity. Proof of concept of this new analytical approach was performed using a thrombin-inhibitor model. The results showed that the qSEC-LC/MS could be developed into an effective affinity-based analytical technique, despite a few limitations such as difficulty in determining the K(d) value accurately.
Discovery Screening Center, Mitsubishi Tanabe Pharma, Saitama 335-8505, Japan. arai.tomonori@me.mt-pharma.co.jp
Tomonori
T
Arai
Masayoshi
M
Uehata
Hiroyuki
H
Akatsuka
Tsutomu
T
Kamiyama
D000818:Animals; D000991:Antithrombins / Q000378:metabolism / Q000494:pharmacology; D001120:Arginine / Q000031:analogs & derivatives; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D020128:Inhibitory Concentration 50; D013058:Mass Spectrometry / Q000379:methods*; D010875:Pipecolic Acids / Q000378:metabolism / Q000494:pharmacology; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D015203:Reproducibility of Results; D013449:Sulfonamides; D013917:Thrombin / Q000032:analysis / Q000378:metabolism
D000991:Antithrombins; D010875:Pipecolic Acids; D011506:Proteins; D013449:Sulfonamides; D001120:Arginine; D013917:Thrombin; C031942:argatroban