Assessing compound binding to the Eg5 motor domain using a thermal shift assay.
Eg5 is a kinesin whose inhibition leads to cycle arrest during mitosis, making it a potential therapeutic target in cancers. Circular dichroism and isothermal titration calorimetry of our pyrrolotriazine-4-one series of inhibitors with Eg5 motor domain revealed enhanced binding in the presence of adenosine 5'-diphosphate (ADP). Using this information, we studied the interaction of this series with ADP-Eg5 complexes using a thermal shift assay. We measured up to a 7 degrees C increase in the thermal melting (T(m)) of Eg5 for an inhibitor that produced IC(50) values of 60 and 130 nM in microtubule-dependent adenosine triphosphatase (ATPase) and cell-based cytotoxicity assays, respectively. In general, the inhibitor potency of the pyrrolotriazine-4-one series in in vitro biological assays correlated with the magnitude of the thermal stability enhancement of ADP-Eg5. The thermal shift assay also confirmed direct binding of Eg5 inhibitors identified in a high-throughput screen and demonstrated that the thermal shift assay is applicable to a range of chemotypes and can be useful in evaluating both potent (nM) and relatively weakly binding (microM) leads. Overall, the thermal shift assay was found to be an excellent biophysical method for evaluating direct binding of a large number of compounds to Eg5, and it complemented the catalytic assay screens by providing an alternative determination of inhibitor potency.
McDonnell
Patricia A
PA
Bristol-Myers Squibb Research and Development, Princeton, NJ 08543, USA. patricia.mcdonnell@bms.com
Yanchunas
Joseph
J
Newitt
John A
JA
Tao
Li
L
Kiefer
Susan E
SE
Ortega
Marie
M
Kut
Stephanie
S
Burford
Neil
N
Goldfarb
Valentina
V
Duke
Gerald J
GJ
Shen
Henry
H
Metzler
William
W
Doyle
Michael
M
Chen
Zhong
Z
Tarby
Christine
C
Borzilleri
Robert
R
Vaccaro
Wayne
W
Gottardis
Marco
M
Lu
Songfeng
S
Crews
Donald
D
Kim
Kyoung
K
Lombardo
Louis
L
Roussell
Deborah L
DL
D000244:Adenosine Diphosphate / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D055592:Biophysical Phenomena; D002151:Calorimetry; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011758:Pyrroles / Q000032:analysis* / Q000737:chemistry*; D013696:Temperature; D014227:Triazines / Q000032:analysis* / Q000737:chemistry*
C470243:KIF11 protein, human; D011758:Pyrroles; D014227:Triazines; C543493:pyrrolotriazine-4-one; D000244:Adenosine Diphosphate; D016547:Kinesins