Driving affinity selection by centrifugal force.
We describe a new approach to affinity selection based on the application of centrifugal force to macromolecules in solution. The method relies on the well known macromolecular hydrodynamic principles of centrifugation. It can be automated and operated in a centralized fashion, or it can be decentralized and used by single researchers or networks of researchers with a minimal additional capital investment. In this method, a centrifugal driving force is used to establish a differential and selective concentration gradient between a therapeutic target and potential ligands in compound libraries. This concentration gradient, in turn, drives the binding of ligands. Once formed, the differential concentration gradient of target macromolecules and ligands is fractionated to capture the self-sorting binding events. Ligand binding is defined by the individual ligand binding constants, so tight binding ligands will essentially distribute identically with the protein target, and weaker binding ligands will not. The level of affinity needed to operationally define tight binding can be adjusted by selecting the initial concentration conditions or centrifugal force. A variety of rapid, commonly available, detection methods can be used to assess binding in the fractionated samples. The method can be broadly applied in drug discovery efforts to examine most types of cell-cell, protein-protein, and protein-small molecule interactions. We describe the application of this method to systems of small molecule interactions with several macromolecules of therapeutic interest.
Harlan
J E
JE
Protein Biochemistry, Global Pharmaceutical Discovery, Abbott Laboratories, Abbott Park, IL 60064, USA.
Egan
D A
DA
Ladror
U S
US
Snyder
S
S
Tang
M I
MI
Buko
A
A
Holzman
T F
TF
D001331:Automation / Q000191:economics / Q000379:methods; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification; D002498:Centrifugation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D008024:Ligands; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
D002352:Carrier Proteins; D008024:Ligands; D046911:Macromolecular Substances; D011506:Proteins