Inhibiting protein-protein interactions: A model for antagonist design

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 (sclE3) against hIL-1 beta that exhibited the equivalent affinity of the soluble IL-1 receptor type I (sIL-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 the sIL-1R in that it failed to bind to either murine IL-1 beta or human/murine 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 other surface plasmon resonance and isothermal titration calorimetry measurements, shows that sclE3 can achieve comparable affinity of binding hIL-1 beta 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.