ATP-Competitive MLKL Binders Have No Functional Impact on Necroptosis.
MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases. A type II kinase inhibitor (cpd 1) has been described that reportedly binds to the MLKL pseudokinase domain and prevents necroptosis. Here we describe five compounds that bind to the MLKL ATP-binding site, however the four MLKL-selective compounds have no activity in rescuing cells from necroptosis. We use kinase selectivity panels, crystallography and a new conformationally sensitive method of measuring protein conformational changes (SHG) to confirm that the one previously reported compound that can rescue cells (cpd 1) is a non-selective type II inhibitor that also inhibits the upstream kinase RIPK1. Although this compound can shift the GFE motif of the activation loop to an "out" position, the accessibility of the key residue Ser358 in the MLKL activation loop is not affected by compound binding to the MLKL active site. Our studies indicate that an ATP-pocket inhibitor of the MLKL pseudokinase domain does not have any impact on the necroptosis pathway, which is contrary to a previously reported study.
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
Bin
B
Ma
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
Doug
D
Marcotte
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
Murugan
M
Paramasivam
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
Klaus
K
Michelsen
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
Ti
T
Wang
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
Andrea
A
Bertolotti-Ciarlet
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
John Howard
JH
Jones
Biodesy Inc., South San Francisco, CA, 94080, United States of America.
Ben
B
Moree
Biodesy Inc., South San Francisco, CA, 94080, United States of America.
Margaret
M
Butko
Biodesy Inc., South San Francisco, CA, 94080, United States of America.
Joshua
J
Salafsky
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
Xin
X
Sun
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
Timothy
T
McKee
Drug Discovery, Biogen Inc., Cambridge, MA, 02142, United States of America.
Laura F
http://orcid.org/0000-0002-5299-5201
LF
Silvian
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects*; D018360:Crystallography, X-Ray; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008958:Models, Molecular; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism
D018841:HSP90 Heat-Shock Proteins; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; C570693:MLKL protein, human; D011494:Protein Kinases; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases