Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85alpha-p110alpha fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors.
Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kalpha pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85alpha-p110alpha fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site.
Sinnamon
Robert H
RH
Biological Reagents and Assay Development Department, Molecular Discovery Research, GlaxoSmithKline, Upper Providence, 1250 South Collegeville Rd., Collegeville, PA 19426, USA. Robert_H_Sinnamon@gsk.com
McDevitt
Patrick
P
Pietrak
Beth L
BL
Leydon
Vaughan R
VR
Xue
Yu
Y
Lehr
Ruth
R
Qi
Hongwei
H
Burns
Matthew
M
Elkins
Patricia
P
Ward
Paris
P
Vincentini
Giorgia
G
Fisher
Donald
D
Grimes
Maggie
M
Brandt
Martin
M
Auger
Kurt R
KR
Ho
Thau
T
Johanson
Kyung
K
Jones
Christopher S
CS
Schwartz
Benjamin
B
Sweitzer
Thomas D
TD
Kirkpatrick
Robert B
RB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019677:Artificial Gene Fusion; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D058541:Class II Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000235:genetics; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D014961:X-Ray Diffraction
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D058541:Class II Phosphatidylinositol 3-Kinases; D058543:Class Ia Phosphatidylinositol 3-Kinase