Inhibiting Protein−Protein Interactions:  A Model for Antagonist Design

Abstract

Protein-protein interactions (PPI) are a ubiquitous mode of transmitting signals in cells and tissues. We are testing a stepwise, generic, structure-driven approach for finding low molecular weight inhibitors of protein-protein interactions. The approach requires development of a high-affinity, single chain antibody directed specifically against the interaction surface of one of the proteins to obtain structural information on the interface. To this end, we developed a single chain antibody (sc1E3) against hIL-1beta that exhibited the equivalent affinity of the soluble IL-1 receptor type I (sIL-1R) for hIL-1beta and competitively blocked the sIL-1R from binding to the cytokine. The antibody proved to be more specific for hIL-1beta than the sIL-1R in that it failed to bind to either murine IL-1beta or human/murine IL-1alpha proteins. Additionally, failure of sc1E3 to bind to several hIL-1beta mutant proteins, altered at receptor site B, indicated that the antibody interacted preferentially with this site. This, coupled with other surface plasmon resonance and isothermal titration calorimetry measurements, shows that sc1E3 can achieve comparable affinity of binding hIL-1beta as the receptor through interactions at a smaller interface. This stable single chain antibody based heterodimer has simplified the complexity of the IL-1/IL-1R PPI system and will facilitate the design of the low molecular weight inhibitors of this interaction.

Topological Frustration and the Folding of Interleukin-1β

“…Interestingly, the residues within and contacting the functionally significant b-bulge in IL-1b 36,37 seem to be the first set of contacts that do not form unless the C-term and N-term of the protein backtrack (see Figure 7). The bulge region is directly involved in receptor binding and signaling in the cell.…”

“…36,37 To analyze the behavior of IL-1b, we partition the protein contacts into different parts. In the following sections, we call the contacts in the N-terminal trefoil region, the N-term, those in the C-terminal trefoil region, the C-term and those in the central trefoil region the center.…”

Long-range Coupling Between Separate Docking Sites in Interleukin-1β

“…Mutation of these residues results in the inability of the antibody to bind the mutant protein. 12 HSQC spectra (Figure 4) of all the mutant proteins compared to the spectrum for WT protein indicate that the global fold is intact. However, there are both local and distal environmental and structural changes upon mutation; dihedral coupling constants derived from the HNHA data (see Results) demonstrate changes near and far from the point of mutation.…”

“…Receptor binding sites have been classified into two regions on IL-1b by mutagenesis, and are designated as site A and site B (Figure 1). 12,13 Interactions at site A involve the side of the cytokine b-barrel that contacts domains 1 and 2 of the receptor, while the interactions at site B involve the open end of the b-barrel and contact receptor domain 3, and the long linker between domains 2 and 3. The lone histidine residue located at position 30 on IL-1b is situated at the end of b-strand 3 in a loop before the 3 10 helix (residues 33-39).…”

Protein‐protein interactions as a target for drugs in proteomics

“…It requires the development of a single chain antibody against one interaction surface of one protein and use of this antibody as a template for design of inhibitors [161]. Recently, several compounds that may act as inhibitors of dimerization and as dimerizers were found.…”