Andrew C. Braisted

Small-Molecule Inhibition of TNF-α

We have identified a small-molecule inhibitor of tumor necrosis factor α (TNF-α) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-α activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-α subunits.

Binding of small molecules to an adaptive protein–protein interface

Understanding binding properties at protein–protein interfaces has been limited to structural and mutational analyses of natural binding partners or small peptides identified by phage display. Here, we present a high-resolution analysis of a nonpeptidyl small molecule, previously discovered by medicinal chemistry [Tilley, J. W., et al. (1997) J. Am. Chem. Soc. 119, 7589–7590], which binds to the cytokine IL-2. The small molecule binds to the same site that binds the IL-2 α receptor and buries into a groove not seen in the free structure of IL-2. Comparison of the bound and several free structures shows this site to be composed of two subsites: one is rigid, and the other is highly adaptive. Thermodynamic data suggest the energy barriers between these conformations are low. The subsites were dissected by using a site-directed screening method called tethering, in which small fragments were captured by disulfide interchange with cysteines introduced into IL-2 around these subsites. X-ray structures with the tethered fragments show that the subsite-binding interactions are similar to those observed with the original small molecule. Moreover, the adaptive subsite tethered many more compounds than did the rigid one. Thus, the adaptive nature of a protein–protein interface provides sites for small molecules to bind and underscores the challenge of applying structure-based design strategies that cannot accurately predict a dynamic protein surface.

Synthesis of proteins by subtiligase.

Application of protein engineering strategies to the redesign of the active site of subtilisin has successfully generated an efficient peptide ligase, subtiligase. The novel enzyme subtiligase has been shown to have many uses, from the total synthesis of RNase A to the semisynthesis of a variety of other proteins. Although the enzyme is in an early stage of development, it shows great promise. Subtiligase will certainly be a useful and important addition to the available strategies for the synthesis of proteins via segment condensation.

Drug discovery at signaling interfaces.

Protein-protein interfaces remain among the most challenging if not intractable targets for small-molecule drug discovery. Despite the wealth of structural information that has accumulated about these complexes and their tremendous value as biotherapeutics, little progress has been made toward identifying small molecules that can specifically target these interfaces. Here, I will review some of the progress we have made in targeting the cytokine receptor interfaces for drug discovery.