Journal: bioRxiv

Article Title: Small-molecule inhibition of Lats kinases promotes Yap-dependent proliferation in postmitotic mammalian tissues

doi: 10.1101/2020.02.11.944157

Figure Lengend Snippet: (A) Protein immunoblotting discloses that treatment of MCF 10A cells with TRULI leaves Hippo signaling intact through the activation of Lats kinases, but that Yap phosphorylation is greatly diminished. (B) Serum starvation of HEK293A cells in the presence of TRULI also demonstrates Lats activation but suppression of Yap phosphorylation. (C) TRULI comprises a thiazolimine backbone with two substituents: a benzyl group and a 7-azaindole hinge-binding motif. (D) In a simulated structure of TRULI in the predicted ATP-binding site of Lats1, the protein is displayed as a ribbon with heavy atoms in atomcolored wire representation with grey carbons. TRULI is displayed in an atom-colored, ball-and-stick representation. Aspartate 846 is positioned to bind the thiazolimine group and phenylalanine 1039 to interact with the benzyl moiety. (E) An in vitro assay of Lats1 kinase activity shows that the IC 50 for TRULI increases with the ATP concentration, from 0.2 nM at 10 μM, to 4.3 nM at 50 μM, and to 80 nM at 250 μM, demonstrating that the compound is an ATP-competitive inhibitor. (F) A kinase assay conducted with serum-starved HEK293A cells indicates an EC 50 of 510 nM for TRULI. Starvation does not significantly deplete the total amount of Yap. In panels E and F, error bars represent SEs. See also Figures S3 and S4.

Article Snippet: The in vitro kinase assay (HTRF KinEASE-STK S1, CisBio 62ST1PEB) was optimized to the linear reaction range of the enzymes Lats1 (Carna 01 −123) and Lats2 (Carna 01-124).

Techniques: Western Blot, Activation Assay, Binding Assay, In Vitro, Activity Assay, Concentration Assay, Kinase Assay