Kinase drug discovery by affinity selection/mass spectrometry (ASMS): application to DNA damage checkpoint kinase Chk1.
Kinase enzymes are involved in a vast array of biological processes associated with human disease; therefore, selective kinase inhibition by small molecules and therapeutic antibodies is an area of intense study. The authors show that drug candidates with immediate value for biological preclinical evaluation can be identified directly through ultra-efficient affinity screening of kinase enzymes and random compound mixtures. The screening process comprises sampling and trapping equilibrium binding between candidate ligands and protein in solution, followed by removal of unbound ligands via 3 rounds of ultrafiltration and direct identification of bound ligands by mass spectrometry. Evaluation of significant peaks is facilitated by automated integration and collation of the mass spectral data and import into custom software for analysis. One Chk1-selective ligand found by using this process is presented in detail. The compound is potent in both enzymatic and Chk1-dependent cellular assays, and specific contacts in the Chk1 active site are shown by X-ray crystallography.
Comess
Kenneth M
KM
Department of Target and Lead Discovery, Global Pharmaceutical R& D, Abbott Laboratories, Illinois 60064-6217, USA.
Trumbull
Jonathan D
JD
Park
Chang
C
Chen
Zehan
Z
Judge
Russell A
RA
Voorbach
Martin J
MJ
Coen
Michael
M
Gao
Lan
L
Tang
Hua
H
Kovar
Peter
P
Cheng
Xueheng
X
Schurdak
Mark E
ME
Zhang
Haiying
H
Sowin
Tom
T
Burns
David J
DJ
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004317:Doxorubicin / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*
D008024:Ligands; D047428:Protein Kinase Inhibitors; D004317:Doxorubicin; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1