Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4.
Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.
Merck Research Laboratory, 33 Avenue Louis Pasteur, Boston, MA 02215, USA. charles.whitehurst@merck.com
Charles E
CE
Whitehurst
Zhiping
Z
Yao
Denise
D
Murphy
Mingxuan
M
Zhang
Shane
S
Taremi
Lisa
L
Wojcik
Julie M
JM
Strizki
Jack D
JD
Bracken
Cliff C
CC
Cheng
Xianshu
X
Yang
Gerald W
GW
Shipps
Michael
M
Ziebell
Elliott
E
Nickbarg
D045744:Cell Line, Tumor; D054377:Chemokine CXCL12 / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*
D054377:Chemokine CXCL12; D008024:Ligands; D019718:Receptors, CXCR4