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.
Subject(s)
Biochemistry/methods , Kinesins/chemistry , Pyrroles/analysis , Pyrroles/chemistry , Triazines/analysis , Triazines/chemistry , Adenosine Diphosphate/metabolism , Biophysical Phenomena , Calorimetry , Cell Line, Tumor , Circular Dichroism , Humans , Kinesins/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Binding , Protein Denaturation , Protein Folding , Protein Structure, Tertiary , TemperatureABSTRACT
Synthesis and SAR of substituted pyrrolotriazine-4-one analogues as Eg5 inhibitors are described. Many of these analogues displayed potent inhibitory activities in the Eg5 ATPase and A2780 cell proliferation assays. In addition, pyrrolotriazine-4-one analogue 26 demonstrated in vivo efficacy in an iv P388 murine leukemia model. Both NMR and X-ray crystallographic studies revealed that these analogues bind to an allosteric site on the Eg5 protein.
Subject(s)
Kinesins/antagonists & inhibitors , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Humans , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Pyrroles/chemistry , Structure-Activity RelationshipABSTRACT
In a high-throughput screening effort, a series of tetrahydroisoquinolines was identified as modest inhibitors of human Eg5. A medicinal chemistry optimization effort led to the identification of R-4-(3-hydroxyphenyl)-N,N-7,8-tetramethyl-3,4-dihydroisoquinoline-2(1H)-carboxamide (32a) as a potent inhibitor of human Eg5 (ATPase IC50 104 nM) with good anti-proliferative activity in A2780 cells (IC50 234 nM).
