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.

Previous studies demonstrated that the naturally occurring electrophile and PPARgamma ligand, nitrolinoleic acid (NO(2)-LA), exists as a mixture of four regioisomers [Alexander, R. L., et al. (2006) Biochemistry 45, 7889-7896]. We hypothesized that these alternative isomers have distinct bioactivities; therefore, to determine if the regioisomers are quantitatively or qualitatively different with respect to PPARgamma activation, NO(2)-LA was separated into three fractions which were identified by NMR (13-NO(2)-LA, 12-NO(2)-LA, and a mixture of 9- and 10-NO(2)-LA) and characterized for PPARgamma interactions. A competition radioligand binding assay showed that all three NO(2)-LA fractions had similar binding affinities for PPARgamma (IC(50) = 0.41-0.60 microM) that were comparable to that of the pharmaceutical ligand, rosiglitazone (IC(50) = 0.25 microM). However, when PPARgamma-dependent transcription activation was examined, there were significant differences observed among the NO(2)-LA fractions. Each isomer behaved as a partial agonist in this reporter gene assay; however, the 12-NO(2) derivative was the most potent with respect to maximum activation of PPARgamma and an EC(50) of 0.045 microM (compare with the rosiglitazone EC(50) of 0.067 microM), while the 13-NO(2) and 9- and 10-NO(2) derivatives were considerably less effective with EC(50) values of 0.41-0.62 microM. We conclude that the regioisomers of NO(2)-LA are not functionally equivalent. The 12-NO(2) derivative appears to be the most potent in PPARgamma-dependent transcription activation, whereas the weaker PPARgamma agonists, 13-NO(2) and 9- and 10-NO(2), may be relatively more important in signaling via other, PPARgamma-independent pathways in which this family of nitrolipid electrophiles is implicated.

Nitrated fatty acids act as endogenous peroxisome proliferator-activated receptor γ (PPARγ) ligands and nitric oxide (NO) donors. We describe the first specific preparation of the two regioisomers of nitrooleic acid, (E)-9-nitrooctadec-9-enoic acid (1) and (E)-10-nitrooctadec-9-enoic acid (2) from cis-cyclooctene and mono-methyl azelate, respectively. These syntheses rely upon a Henry condensation between a nine-carbon nitro component and a nine-carbon aldehyde. Preliminary chemiluminescence NO detection studies reveal the ability of these nitrated fatty acids to release NO.Nitrated fatty acids have emerged as a unique class of endogenously produced signaling molecules. Initial studies reveal that nitrated derivatives of linoleic acid (18:2) occur in concentrations of ~500 nM in human red blood cells and plasma making nitrated fatty acids the single largest pool of bioactive nitrogen oxides in the vasculature. 1 Additional studies show that nitrolinoleate (LNO 2 ) acts as a ligand of the peroxisome proliferator-activated receptor γ (PPARγ). 2 Activation of this transcription factor results in gene expression that affects numerous critical cellular processes including growth and differentiation, inflammation, metabolic homeostasis and vasomotor processes. 3 Other studies show that LNO 2 also spontaneously releases nitric oxide (NO), an endogenous free radical signaling mediator that regulates a number of biological processes including blood pressure, neurotransmission, and platelet aggregation. 4,5 These interesting molecules thus bridge fatty acid-derived signaling with nitric oxide-mediated signaling. More recent work shows that (E)-9-and (E)-10-nitrooctadec-9-enoic acids (nitrated oleic acids, OA-NO 2 ) occur with a Correspondence to: S. Bruce King, kingsb@wfu.edu. Supporting Information Available: Full experimental details for the preparation of all intermediates and products, as well as complete spectroscopic and analytical data for all characterized compounds. Copies of 1 H and 13 C NMR spectra for 1, 2, 4, 6, 7, 12, and 13 are also included. This material is available free of charge via the Internet at http://pubs.acs.org. Current methods for the synthesis of these nitrated fatty acids involve the modification of previous nitroselenylation strategies used to synthesize conjugated nitroalkenes. 7,8 Alternatively, addition of nitronium tetrafluoroborate to an unsaturated fatty acid or fatty acid hydroperoxide also forms nitrated fatty acids. 9 These non-specific procedures form regioisomeric mixtures of nitro alkene products that require multiple purification steps. More importantly, these non-specific syntheses prevent strict analysis of the structural requirements for the biological activity of these unique molecules. Here we report the first regio-and stereospecific syntheses of (E)-9-nitro-octadec-9-enoic acid (1) and (E)-10-nitrooctadec-9-enoic acid (2) and show that these nitrated lipids spontaneously release nitric oxide. NIH Public AccessScheme 1 describes the retrosynthetic strategy for ...

Nitroalkene fatty acids are potent endogenous ligand activators of PPARγ-dependent transcription. Previous studies with the naturally occurring regioisomers of nitrolinoleic acid revealed that the isomers are not equivalent with respect to PPARγ activation. To gain further insight into the structureactivity relationships between nitroalkenes and PPARγ, we examined additional naturally occurring nitroalkenes derived from oleic acid, 9-nitrooleic acid (E-9-NO 2 -18:1 [1]) and 10-nitrooleic acid (E-10-NO 2 -18:1 [2]), and several synthetic nitrated enoic fatty acids of variable carbon chain length, double bonds, and nitration site. At submicromolar concentrations, E-12-NO 2 derivatives were considerably more potent than isomers nitrated at carbons 5, 6, 9, 10 or 13; and, chain length (16 versus 18) or number of double bonds (one versus two) was of little consequence for PPARγ activation. Interestingly, at higher concentrations (> 2 μM) the nitrated enoic fatty acids (E-9-NO 2 -18:1 [1], E-9-NO 2 -16:1 [3], E-10-NO 2 -18:1 [2], and E-12-NO 2 -18:1 [7]) deviated significantly from the saturable pattern of PPARγ activation observed for nitrated 1,4-dienoic fatty acids

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