Structural information is a pillar of modern drug discovery, especially so with fragments. Consistent with our philosophy of orthogonality, ZoBio is unique in leveraging the synergy of NMR and X-ray crystallography to provide structural information earlier and more robustly to highly challenging targets.
Crystallography and NMR are complementary in many ways. For one, NMR is performed on protein in solution instead of the crystal lattice. For another, crystallography typically either provides an unambiguous structure or not, NMR can provide structural information at increasing levels of resolution. Together, these very different technologies make each other better.
Early in any drug discovery campaign, the binding site of interesting compounds is often unknown, particularly for weakly binding fragments or analogs. In such cases, interpreting the electron density derived from even high resolution diffraction data may be difficult due to the uncertainty in both location and occupancy of the ligand. We have used simple ligand observed NMR experiments to both independently confirm binding of the compound to the target and define the binding site by adding a known “competing” molecule. This extra level of information allows us to confidently assign the correct electron density to the small molecule and determine the pose without requiring stable isotope labeling of the target.
This tight integration of crystallography, NMR and protein engineering allows efficient exploration of many combinations of protein constructs, solution conditions and crystallization conditions. This increases the success rate in generating robust crystals with sufficient diffraction power. We routinely implement hybrid schemes of co-crystallization and soaking to maximize occupancy of the ligand. In order to maximize our “shots on goal”, we also have developed an approach to efficiently screen up to 24 compounds per batch of crystals which often includes multiple members of an interesting chemotype. We can also use in situ diffraction techniques to rapidly screen through up to 100 crystals at a time to select those that are most interesting for collection of a complete data set.
Although the priority, crystallography does not always deliver the necessary structural information. In these cases we can use our unique expertise to efficiently obtain structural constraints from NMR. ZoBio has contributed to automated resonance assignment  which has revolutionized the time required, reducing it from many months to as little as a couple of weeks. With this information, the binding site can be readily mapped by titrating a compound and observing selective perturbations to the NMR spectrum of the protein. Such studies can be conveniently performed on batches of ~10 compounds in 1 week. Of course, where necessary, the 3D structure of the complex can be elucidated using NMR, often through use of the crystal structure(s) of apo protein, thereby completing the circle of synergy.
 – Pritisanac et al., J. Am. Chem. Soc., 2017, v.139, p.9523-9533