title: Assessing compound binding to the Eg5 motor domain using a thermal shift assay.
abstract: 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.
authors.affiliation: Bristol-Myers Squibb Research and Development, Princeton, NJ 08543, USA. patricia.mcdonnell@bms.com
authors.forename: Patricia A
authors.initials: PA
authors.lastname: McDonnell
authors.forename: Joseph
authors.initials: J
authors.lastname: Yanchunas
authors.forename: John A
authors.initials: JA
authors.lastname: Newitt
authors.forename: Li
authors.initials: L
authors.lastname: Tao
authors.forename: Susan E
authors.initials: SE
authors.lastname: Kiefer
authors.forename: Marie
authors.initials: M
authors.lastname: Ortega
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kut
authors.forename: Neil
authors.initials: N
authors.lastname: Burford
authors.forename: Valentina
authors.initials: V
authors.lastname: Goldfarb
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Duke
authors.forename: Henry
authors.initials: H
authors.lastname: Shen
authors.forename: William
authors.initials: W
authors.lastname: Metzler
authors.forename: Michael
authors.initials: M
authors.lastname: Doyle
authors.forename: Zhong
authors.initials: Z
authors.lastname: Chen
authors.forename: Christine
authors.initials: C
authors.lastname: Tarby
authors.forename: Robert
authors.initials: R
authors.lastname: Borzilleri
authors.forename: Wayne
authors.initials: W
authors.lastname: Vaccaro
authors.forename: Marco
authors.initials: M
authors.lastname: Gottardis
authors.forename: Songfeng
authors.initials: S
authors.lastname: Lu
authors.forename: Donald
authors.initials: D
authors.lastname: Crews
authors.forename: Kyoung
authors.initials: K
authors.lastname: Kim
authors.forename: Louis
authors.initials: L
authors.lastname: Lombardo
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Roussell
mesh_terms: 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*
chemical_list: C470243:KIF11 protein, human; D011758:Pyrroles; D014227:Triazines; C543493:pyrrolotriazine-4-one; D000244:Adenosine Diphosphate; D016547:Kinesins