As part of an international team, led by HepaRegeniX, ZoBio solved the structure of an inhibitor bound to the kinase MKK4 using a non-classical approach to structural elucidation with NMR. These efforts have recently been described in Cell detailing the preclinical characterization and development of the clinical candidate HRX215, a first-in-class small molecule inhibitor of MKK4. 

Mitogen-Activated Protein (MAP) Kinase Kinase 4 (MKK4) is a dual specific MAP2 kinase that was recently shown to be a master regulator of liver cell (hepatocyte) regeneration. Modulation of MKK4 activity therefore has profound therapeutic implication for patients suffering from chronic or acute liver disease. Including cirrhosis of the liver, primary liver tumors and cancer metastasized to the liver.  

Initial efforts to obtain structural information for the inhibitor bound MKK4 kinase domain were focused on X-ray crystallography. While a structure was obtained, the resolution was insufficient to resolve the binding mode of the inhibitor. To support structure-based inhibitor design, the strategic shift was made to NMR. When conventional NMR methods yielded insufficient spectral quality, a close collaboration between the NMR spectroscopists and protein scientists at ZoBio enabled the use of a non-classical approach to NMR structural elucidation. 

The method employed to finally resolve the inhibitor-bound MKK4 structure was based on selective labeling of methyl groups in the protein. To assign peaks and validate assignments, these sparse spectral data were supplemented with the structural data of a close homolog and multiple rounds of point-mutant design. Together, these experiments resulted in the unambiguous resolution of the structure of the MKK4-inhibitor complex.  

 This study underscores the versatility of the techniques and expertise within ZoBio and demonstrates the strength of close collaboration among our scientists in solving challenging problems!