Inhibiting protein-protein interactions: A model for antagonist design

Protein-protein interactions (PPI) are a ubiquitous mode of transmitting si gnals in cells and tissues. We are testing a stepwise, generic, structure-d riven approach for finding low molecular weight inhibitors of protein-prote in interactions. The approach requires development of a high-affinity, sing le chain antibody directed specifically against the interaction surface of one of the proteins to obtain structural information on the interface. To t his end, we developed a single chain antibody (sclE3) against hIL-1 beta th at exhibited the equivalent affinity of the soluble IL-1 receptor type I (s IL-1R) for hIL-1 beta and competitively blocked the sIL-1R from binding to the cytokine. The antibody proved to be more specific for hIL-1 beta than t he sIL-1R in that it failed to bind to either murine IL-1 beta or human/mur ine IL-1 alpha proteins. Additionally, failure of sc 1E3 to bind to several hIL-1 beta mutant proteins, altered at receptor site B, indicated that the antibody interacted preferentially with this site. This, coupled with othe r surface plasmon resonance and isothermal titration calorimetry measuremen ts, shows that sclE3 can achieve comparable affinity of binding hIL-1 beta as the receptor through interactions at a smaller interface. This stable si ngle 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 w eight inhibitors of this interaction.