ABSTRACT
The two subunits of core binding factor (Runx1 and CBFbeta) play critical roles in hematopoiesis and are frequent targets of chromosomal translocations found in leukemia. The binding of the CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein to Runx1 is essential for leukemogenesis, making this a viable target for treatment. We have developed inhibitors with low micromolar affinity which effectively block binding of Runx1 to CBFbeta. NMR-based docking shows that these compounds bind to CBFbeta at a site displaced from the binding interface for Runx1, that is, these compounds function as allosteric inhibitors of this protein-protein interaction, a potentially generalizable approach. Treatment of the human leukemia cell line ME-1 with these compounds shows decreased proliferation, indicating these are good candidates for further development.
Subject(s)
Allosteric Site , Cell Proliferation/drug effects , Core Binding Factor Alpha 2 Subunit/antagonists & inhibitors , Core Binding Factor beta Subunit/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Leukemia/pathology , Binding Sites , Cell Line, Tumor , Core Binding Factor Alpha 2 Subunit/chemistry , Core Binding Factor Alpha 2 Subunit/metabolism , Core Binding Factor beta Subunit/chemistry , Core Binding Factor beta Subunit/metabolism , Enzyme Inhibitors/chemistry , Fluorescence Resonance Energy Transfer , Hematopoiesis/genetics , Hematopoiesis/physiology , Humans , Leukemia/metabolism , Magnetic Resonance Spectroscopy , Myosin Heavy Chains/chemistry , Myosin Heavy Chains/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Smooth Muscle Myosins/chemistry , Smooth Muscle Myosins/metabolism , Translocation, Genetic/genetics , Translocation, Genetic/physiologyABSTRACT
The Runt domain is the DNA binding domain of the core binding factor (CBF) Runx subunits. The CBFs are transcription factors that play critical roles in hematopoiesis, bone, and neuron development in mammals. A common non-DNA binding CBFbeta subunit heterodimerizes with the Runt domain of the Runx proteins and allosterically regulates its affinity for DNA. Previous NMR dynamics studies suggested a model whereby CBFbeta allosterically regulates DNA binding by quenching conformational exchange in the Runt domain, particularly in the S-switch region and the betaE'-F loop. We sought to test this model, and to this end introduced all possible single amino acid substitutions into the S-switch region and the betaE'-F loop, and screened for mutations that enhanced DNA-binding. We demonstrate that one Runt domain mutant, R164N, binds both DNA and CBFbeta with higher affinity, but it is less sensitive to allosteric regulation by CBFbeta. Analysis of NMR relaxation data shows that the chemical exchange exhibited by the wild-type Runt domain is largely quenched by the R164N substitution. These data support a model in which the dynamic behavior of a network of residues connecting the CBFbeta and DNA binding sites on the Runt domain plays a critical role in the mechanism of allosteric regulation. This study provides an important functional link between dynamic behavior and protein allosteric function, consistent with results on other allosterically regulated proteins.
