A Protoberberine derivative HWY336 selectively inhibits MKK4 and MKK7 in mammalian cells: the importance of activation loop on selectivity.
A protoberberine derivative library was used to search for selective inhibitors against kinases of the mitogen-activated protein kinase (MAPK) cascades in mammalian cells. Among kinases in mammalian MAPK pathways, we identified a compound (HWY336) that selectively inhibits kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7). The IC50 of HWY336 was 6 µM for MKK4 and 10 µM for MKK7 in vitro. HWY336 bound to both kinases reversibly via noncovalent interactions, and inhibited their activity by interfering with access of a protein substrate to its binding site. The binding affinity of HWY336 to MKK4 was measured by surface plasmon resonance to determine a dissociation constant (Kd) of 3.2 µM. When mammalian cells were treated with HWY336, MKK4 and MKK7 were selectively inhibited, resulting in inhibition of c-Jun NH2-terminal protein kinases in vivo. The structural model of HWY336 bound to either MKK4 or MKK7 predicted that HWY336 was docked to the activation loop, which is adjacent to the substrate binding site. This model suggested the importance of the activation loop of MKKs in HWY336 selectivity. We verified this model by mutating three critical residues within this loop of MKK4 to the corresponding residues in MKK3. The mutant MKK4 displayed similar kinase activity as wild-type kinase, but its activity was not inhibited by HWY336 compared to wild-type MKK4. We propose that the specific association of HWY336 to the activation loop of MKK4/MKK7 is responsible for its selective inhibition.
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.
Namil
N
Kim
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.
Jeongyeon
J
Park
Department of Bio-New Drug Development, College of Medicine, Chosun University, Gwangju, Korea.
Changdev G
CG
Gadhe
Department of Bio-New Drug Development, College of Medicine, Chosun University, Gwangju, Korea; Department of Cellular Molecular Medicine and Research Center for Resistant Cells, College of Medicine, Chosun University, Gwangju, Korea.
Seung Joo
SJ
Cho
School of Electrical and Electronic Engineering, College of Engineering, Yonsei University, Seoul, Korea.
Youngjin
Y
Oh
School of Electrical and Electronic Engineering, College of Engineering, Yonsei University, Seoul, Korea.
Donghyun
D
Kim
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.
Kiwon
K
Song
D001600:Berberine Alkaloids / Q000494:pharmacology*; D002460:Cell Line; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D048688:MAP Kinase Kinase 7 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D001600:Berberine Alkaloids; C009090:protoberberine; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; C482559:MAP2K4 protein, human; C482569:MAP2K7 protein, human