Abstract

The pim family of kinases are small cytoplasmic serine/threonine kinases implicated in the development of leukemias, lymphomas, and prostate cancer. Expression of pim-1 and pim-2 is induced by several oncogenes and growth factors. These kinases confer a survival phenotype through modulation of the expression or activity of various bcl-2 family members. To identify prototype small molecule inhibitors of pim-1 we have undertaken a series of experimental and computational studies. Central to our efforts has been the development of a homology model of the PIM-1 protein, using death-associated kinase as a template. After a series of computational refinements using the CFF forcefield, we then further modified our model through experimental approaches. The members of the pim family appear to have a unique, conserved structure to the hinge region, based on its length and the presence of two invariant prolines. This observation suggested that ATP-pocket binding compounds could be identified that would have selectivity for pim-1. We used a liquid phase, ELISA-based in vitro kinase assay to measure IC50 values for a set of 14 flavonoids, a family of known kinase inhibitors. Calculated binding energies for the test flavonoids were then compared with measured IC50 values, and the model giving the highest correlations was then used for further studies. A set of 25 additional flavonoids was then examined for binding energy to the homology model. Our studies predicted that the flavonoids quercetagetin and gossypetin could be potential pim-1 inhibitors. This hypothesis was then tested by in vitro kinase assay. Indeed these two flavonoids were found to be active pim-1 inhibitors with high-nanomolar potency. Quercetagetin was found to be competitive with ATP in the kinase assay. Furthermore the two flavonoids were seen to have a degree of selectivity for pim-1 kinase, compared with related serine-threonine kinases and a tyrosine kinase.

IC50 (micromolar) of flavonoids against PIM-1 and other kinases

Inhibitor PIM-1 PIM-2 cAMP-dep. kinase c-ABL 
quercetagetin 0.33 4.0 21 >200 
gossypetin 0.430 7.1 250 >200 
quercetin 2.0 ND ND 5.7 
Inhibitor PIM-1 PIM-2 cAMP-dep. kinase c-ABL 
quercetagetin 0.33 4.0 21 >200 
gossypetin 0.430 7.1 250 >200 
quercetin 2.0 ND ND 5.7 

In contrast the potency and pim-1-selectivity of the related flavonoid quercetin (a promiscuous inhibitor of kinases) was much inferior. SAR analysis of the flavonoid set showed that pim-1 selectivity depended on the presence of three hydrogen bond donors on the flavonoid A ring. Quercetagetin has been tested for biologic effects on factor-dependent FDCP1 cells transfected with a cDNA for human pim-1. These test cells show prolonged, pim-1-dependent survival after removal of IL-3 from the growth medium. This phenotype was completely abolished by treatment with quercetagetin (IC50 = 3-5micromolar final concentration). Flavonoids such as quercetagetin and gossypetin may serve as guides for the development of small molecule inhibitors specific for pim family kinases. Such reagents will be useful for determining the role of constitutive pim-1 expression in the development of leukemias and lymphomas.

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