NMR-driven discovery of benzoylanthranilic acid inhibitors of far upstream element binding protein binding to the human oncogene c-myc promoter.

Reversal of aberrant gene expression that is induced by the proto-oncogene c-myc is likely to be effective for treating a variety of tumors that rely on this pathway for growth. One strategy to down-regulate the c-myc pathway is to target transcription factors that regulate its own expression. A host of proteins act in coordination to regulate c-myc expression and any one of them are theoretical targets for small-molecule therapy. Experimentally, it has been shown that the far upstream element (FUSE) binding protein (FBP) is essential for c-myc expression, and reductions in FBP levels both reduce c-myc expression and correlate with slower cell growth. FBP binds to ssDNA by capturing exposed DNA bases in a hydrophobic pocket. This suggests that a small molecule could be designed to occupy this pocket and inhibit FBP function. Using a variety of screening methodologies, we have identified ligands that bind to the DNA binding pockets of the KH domains of FBP. Gel shift analyses using full length FBP and a related transcription factor confirm that a small-molecule lead compound inhibits DNA binding in a specific manner. The benzoylanthranilic acid compounds described here represent leads in the design of FBP inhibitors that can serve as useful tools in the study of c-myc regulation and in the development of therapeutics that target the c-myc pathway.

Discovery of a novel small molecule binding site of human survivin.

Survivin is one of the most tumor-specific genes in the human genome and is an attractive target for cancer therapy. However, small-molecule ligands for survivin have not yet been described. Thus, an interrogation of survivin which could potentially both validate a small-molecule therapy approach, and determine the biochemical nature of any of survivin's functions has not been possible. Here we describe the discovery and characterization of a small molecule binding site on the survivin surface distinct from the Smac peptide-binding site. The new site is located at the dimer interface and exhibits many of the features of highly druggable, biologically relevant protein binding sites. A variety of small hydrophobic compounds were found that bind with moderate affinity to this binding site, from which one lead was developed into a group of compounds with nanomolar affinity. Additionally, a subset of these compounds are adequately water-soluble and cell-permeable. Thus, the structural studies and small molecules described here provide tools that can be used to probe the biochemical role(s) of survivin, and may ultimately serve as a basis for the development of small molecule therapeutics acting via direct or allosteric disruption of binding events related to this poorly understood target.

Site-directed rotational resonance solid-state NMR distance measurements probe structure and mechanism in the transmembrane domain of the serine bacterial chemoreceptor.

The serine receptor of bacterial chemotaxis is an ideal system in which to investigate the molecular mechanism of transmembrane signaling. Solid-state nuclear magnetic resonance (NMR) techniques such as rotational resonance provide a means for measuring local structure and ligand-induced structural changes in intact membrane proteins bound to native membrane vesicles. A general site-directed biosynthetic (13)C labeling strategy is used to direct the distance measurements to a specific site; the distance is measured between a unique Cys residue and a non-unique, low-abundance residue (Tyr or Phe). A (13)C-(13)C internuclear distance measurement from (13)CO(i) to (13)C beta(i + 3) at the periplasmic edge of the second membrane-spanning helix (TM2) of 5.1 +/- 0.2 A is consistent with the predicted alpha-helical structure and thus demonstrates an accurate long-distance rotational resonance measurement in the 120 kDa membrane-bound receptor. These measurements require a correction for the rotational resonance exchange between the multiple labels of the non-unique amino acid and the natural-abundance (13)C, which is critical to distance measurements in complex systems. A second (13)C-(13)C distance measurement between the transmembrane helices provides a high-resolution measurement of tertiary structure in the transmembrane region. The measured 5.0-5.3 A distance in the presence and absence of ligand is consistent with structural models for the transmembrane region and a proposed signaling mechanism in which ligand binding induces a 1.6 A translation of TM2. This approach can be used for additional measurements of the structure of the transmembrane region and to determine whether the ligand-induced motion is indeed propagated through the transmembrane helices.