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.
Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in several developmental pathways, including hematopoiesis and bone development. Mutations in CBF genes are found in leukemias and bone disorders. CBFs consist of a DNA-binding CBF␣ subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBF subunit. CBF␣ binds DNA in a sequence-specific manner, whereas CBF enhances DNA binding by CBF␣. Recent structural analyses of the DNA-binding Runt domain of CBF␣ and the CBF subunit identified the heterodimerization surfaces on each subunit. Here we identify amino acids in CBF that mediate binding to CBF␣. We determine the energy contributed by each of these amino acids to heterodimerization and the importance of these residues for in vivo function. These data refine the structural analyses and further support the hypothesis that CBF enhances DNA binding by inducing a conformational change in the Runt domain.Core-binding factors (CBFs) 1 are a small family of heterodimeric transcription factors that play critical roles in normal development and disease. CBFs consist of two subunits, CBF␣ and CBF. The CBF␣ subunit binds DNA in a sequencespecific manner (1, 2). CBF heterodimerizes with CBF␣ and increases its affinity for DNA (3, 4). The mechanism by which CBF enhances DNA binding by CBF␣ is unusual in that CBF does not establish contacts to additional phosphates or bases in the DNA (1, 4). It has been proposed that CBF stabilizes a conformation of CBF␣ favorable for DNA binding (5-7).CBF␣ subunits are encoded by three related genes, RUNX1, RUNX2, and RUNX3 (formerly CBFA1-3, AML1-3, or Pebpa2a-c) (2,8,9). A single gene, CBFB, encodes the CBF subunit subunit (3, 4). The RUNX2 gene is required for bone formation and is implicated in a human genetic disorder, cleidocranial dysplasia, a disease characterized by moderate skeletal malformations (10 -12). The in vivo function of RUNX3 is unknown. RUNX1 (AML1) and CBFB are required for hematopoiesis and are the most frequent targets of mutations in human leukemias (13)(14)(15)(16)(17). RUNX1 is disrupted by the t(8; 21)(q22;q22) in 15% of de novo acute myeloid leukemias (M2 subtype), the t(12;21)(p13;q22) in 30% of pediatric de novo acute lymphocytic leukemias, and by the t(1;21)(p36;q22), t(3; 21)(q26;q22), t(5;21)(q13;q22), t(12;21)(q24;q22), t(14;21)(q22; q22), t(15;21)(q22;q22), t(16;21)(q24;q22), and t(17;21)(q11.2; q22) in therapy-related leukemias and myelodysplasia (8, 18 -23). Haploinsufficiency of RUNX1 is responsible for a rare familial platelet disorder with propensity for acute myelogenous leukemia (FPD/AML) (24). Biallelic point mutations in the RUNX1 gene are found in AML of the M 0 subtype (25). The CBFB gene is disrupted in approximately 15% of acute myeloid leukemias by inv(16)(p13;q22), t(16;16), and del(16)(q22) (26,27). Together, disruptions of the RUNX2 and CBFB genes account for approximately 25% of all de novo acute leukemias, making them the most frequently disrupted genes in human leukem...
Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFK K subunit and a non-DNA binding CBFL L subunit that increases the affinity of CBFK K for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFK K:L L:DNA complex. Dissociation constants for all four equilibria involving the CBFK K Runt domain, CBFL L, and DNA were defined. Conformational changes associated with interactions between CBFK K, CBFL L, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFL L locks in' a high affinity DNA binding conformation of the CBFK K Runt domain.z 2000 Federation of European Biochemical Societies.
The goal of structural genomics initiatives is to determine complete sets of protein structures that represent recently sequenced genomes. The development of new high throughput methods is an essential aspect of this enterprise. Residue type and sequential assignments obtained from specifically labeled samples, when combined with 3D heteronuclear data, can significantly increase the efficiency and accuracy of the assignment process, the first step in structure determination by NMR. A protocol for the design of specifically labeled samples with high information content is presented along with a description of the experiments used to extract essential information using 2D versions of 3D heteronuclear experiments. In vitro protein synthesis methods were used to produce four specifically labeled samples of the 23.5 kDa protein phosphoserine phosphatase (PSP) from Methanoccous jannaschii (MJ1594). Each sample contained two (13)C/(15)N-labeled amino acids and one (15)N-labeled amino acid. The 135 type and 14 sequential assignments obtained from these samples were used in conjunction with 3D data obtained from uniformly (13)C/(15)N-labeled and (2)H/(13)C/(15)N-labeled protein to manually assign the backbone (1)H(N), (15)N, (13)CO, (13)C(alpha), and (13)C(beta) signals. Using an automated assignment algorithm, 30% more assignments were obtained when the type and sequential assignments were used in the calculations.
A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC-MS/MS technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (K(D)) and PDE10A target density (B(max)) were determined to be 0.94 nM and 2.3 pmol/mg protein, respectively, using [(3)H]5 on rat striatum homogenate. Autoradiography on rat brain sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific binding of [(3)H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats using LC-MS/MS technology.
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