We have discovered a class of PI3Kγ inhibitors exhibiting over 1,000-fold selectivity over PI3Kα and PI3Kβ. On the basis of X-ray crystallography, hydrogen-deuterium exchange–mass spectrometry and surface plasmon resonance experiments we propose that the cyclopropylethyl moiety displaces the DFG motif of the enzyme away from the adenosine tri-phosphate binding site, inducing a large conformational change in both the kinase- and helical domains of PI3Kγ. Site directed mutagenesis explained how the conformational changes occur. Our results suggest that these cyclopropylethyl substituted compounds selectively inhibit the active state of PI3Kγ, which is unique to these compounds and to the PI3Kγ isoform, explaining their excellent potency and unmatched isoform selectivity that were confirmed in cellular systems. This is the first example of a Class I PI3K inhibitor achieving its selectivity by affecting the DFG motif in a manner that bears similarity to DFG in/out for type II protein kinase inhibitors.
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The authors would like to thank A. Gunnarsson, AstraZeneca, for input on membrane interactions, M. McAllister; AstraZeneca for facilitating the project and proofreading the manuscript and N. Bond, MedImmune, for initial HDX–MS experiments. G.G. is a fellow of the AstraZeneca post doc program. R.L.W. acknowledges support by the Medical Research Council (MC_U105184308) and an MRC—AstraZeneca/MedImmune Blue Sky Collaborative Research Grant (MC_A024-5PF9G). M.P.W. was supported by the Swiss Commission for Technology and Innovation; the Stiftung für Krebsbekämpfung Grant 341 and Swiss National Science Foundation Grants 310030_153211 and 316030_133860.
Supplementary Tables 1–5, Supplementary Figures 1–21
The movie based on our crystal structure and HDX-MS observations illustrates our model of how AZ2 binds to PI3Kγ and induces large-scale conformational changes that bring the PI3Kγ into its active conformation, but with the inhibitor bound. The movie was prepared with PyMOL (Schrödinger). In the first 1/3 of the move, AZ2 binds in the ATP-binding pocket. This initiates two conformational transitions that were created by the PyMOL Morph command. In the first transition forming the second 1/3 of the movie, the active site of the basal-state PI3Kγ (represented by the 4FUL PDB structure) 5 with the “Lock” intact morphs into a conformation with the lock broken and the helix kα12 lifted off the catalytic and activation loops, a conformation represented by our structure of p110δ bound to AZ2. We propose that this transition gives rise to the initial catalytically competent state of PI3Kγ. In the second transition, forming the last 1/3 of the movie, helix kα12 undergoes a large conformational change accompanied by widespread changes in conformation of the kinase domain, as the enzyme assumes the fully active conformation capable of carrying out catalysis on the membrane surface. We believe that the binding of this highly specific PI3Kγ inhibitor provides some insight into the conformational rearrangements that accompany the natural process of activation of the enzyme on membranes.