Two systems are currently under study as intramural collaborations. The high-affinity binding of the growth-factor receptor-bound protein 2 (Grb2) SH2 domain to tyrosine-phosphorylated cytosolic domains of receptor tyrosine kinases (RTKs) is an attractive target for therapeutic intervention in many types of cancer. To this end, we have determined the structures of two crystal forms of a complex between the Grb2 SH2 domain and a potent non-phosphorus containing macrocyclic peptide mimetic that exhibits significant anti-proliferative effects against erbB-2-dependent breast cancers. This agent represents a second generation inhibitor with greatly improved binding affinity and bioavailability compared to its open-chain counterpart. The structures were determined at 2.0 and 1.8 with one and two domain-swapped dimers per asymmetric unit, respectively. The mode of binding and specific interactions between the protein and the inhibitor provide insight into the high potency of this class of macrocylic compounds and may aid in further optimization as part of the iterative rational drug design process. We are also involved in a multidisciplinary collaboration to develop potent and specific inhibitors of human Chk2 kinase. Our role in this project is to determine co-crystal structures of Chk2 in complex with small molecule inhibitors. Thus far we have determined 8 co-crystal structures with different compounds and this has provided a wealth of information about the mode of inhibitor binding and led to ideas about how to improve specificity and potency. We have also produced site-specifically biotinylated Chk2 for surface plasmon resonance studies of inhibitor binding. Finally, we have determined the structure of the POZ domain of human LRF, a master regulator of oncogenesis, as a first step toward the development of inhibitors of this protein (see Scientific Advances).
