The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold.
Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.
From the Center for Proteomic Chemistry, sascha.gutmann@novartis.com.
Sascha
S
Gutmann
From the Center for Proteomic Chemistry.
Alexandra
A
Hinniger
From the Center for Proteomic Chemistry.
Gabriele
G
Fendrich
From the Center for Proteomic Chemistry.
Peter
P
Drückes
From the Center for Proteomic Chemistry.
Sylvie
S
Antz
Global Discovery Chemistry, and.
Henri
H
Mattes
Global Discovery Chemistry, and.
Henrik
H
Möbitz
Global Discovery Chemistry, and.
Silvio
S
Ofner
Global Discovery Chemistry, and.
Niko
N
Schmiedeberg
Global Discovery Chemistry, and.
Aleksandar
A
Stojanovic
From the Center for Proteomic Chemistry.
Sebastien
S
Rieffel
From the Center for Proteomic Chemistry.
André
A
Strauss
Global Discovery Chemistry, and.
Thomas
T
Troxler
Global Discovery Chemistry, and.
Ralf
R
Glatthar
Autoimmunity Transplantation Inflammation, Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland.
Helmut
H
Sparrer
D018360:Crystallography, X-Ray; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D020930:MAP Kinase Kinase Kinases; C069937:MAP3K8 protein, human
cancer; drug discovery; immunology; mitogen-activated protein kinase (MAPK); structural biology