Fig 2: Homology-model guides design of covalent-allosteric inhibitors to address Akt isoforms.a General overview of the closed conformation of protein kinase Akt (kinase domain: gray, PH domain: green; based on PDB: 6HHF)36 and the druggable binding pockets (ATP-pocket within kinase domain: gray (PDB: 4GV1)52, allosteric-interdomain pocket: blue (PDB: 6HHF), PIP3-binding pocket within PH domain: gray (PDB 2UVM)53). The variety of the binding site forming residues among the three isoforms are given in percentage; b View of the interdomain binding site in Akt1 (white; PDB 6S9X: with the covalent-allosteric Akt inhibitor 15c (blue))17 in comparison to homology models of Akt2 (red) and Akt3 (yellow). The structural changes within the allosteric site and the different side chain residues of each isoform are highlighted; c Modeling studies with recently found pyrazinonic CAAI 16c into Akt isoform homology model highlight structural differences and possible interactions within the allosteric binding site (Akt1(white) PDB: 6S9X; Akt2 (red) and Akt3 (yellow) homology model)17. The side view shows an enlarged space in Akt2 and Akt3, ideally to allow the binding of the hydroxyl phenyl moiety (Supplementary Fig. 4); d Structure-guided design approach towards a focused set of small molecules for evaluation of Akt isoform-selectivity: trisubstituted, biarylic pyridines function as core scaffold (blue). A benzimidazolone linker (green) is decorated with the Michael acceptor warhead (gray) to address the nucleophilic cysteine residues within the allosteric binding site. Different moieties (red) with various chemical properties and spatial demand should be connected over a defined linkage to the core molecule.

Fig 3: β1 integrins induce Ser473 phosphorylation on both AKT1 and AKT2.Adhesion assays were performed using MCF7 cells and plates coated with Pluronic or invasin followed by immunoprecipitation with isoform-specific AKT1, AKT2 and AKT3 antibodies. (A) The lysates were centrifuged in order to remove the insoluble fraction. The resulting pellet was investigated for the presence of AKT pSer473 by SDS-PAGE and western blotting. A sample of each lysate supernatant after centrifugation was investigated for the presence of AKT pSer473, AKT1 and AKT2 proteins. (B) Immunoprecipitation was performed with antibodies specific for AKT1, AKT2 or AKT3 (included as control) in pre-cleared lysates. The precipitates were boiled and subjected to two parallel SDS-PAGE and western blot analyses in order to detect total AKT or AKT pSer473 protein. In spite of using light-chain specific anti-rabbit-HRP conjugates, some weaker unspecific bands (marked with an *) are visible due to interaction of the primary western antibodies with the heavy chain of the IgGs used for immunoprecipitation.

Fig 4: miR-181a-5p modulates the AKT3-FOXO3-IL-23R/CCL5 pathway in Th17 cells(A and B) Western blot analysis for AKT pathway-associated protein in Th17 cells infected with LV-Ctrl or LV-181a (MOI =50) in vitro (A), and in CD4+ T cells isolated from LV-Ctrl or LV-181a-infected EAU mice (B). Statistical analysis data are shown in the right panel (n = 4 per group).(C) Representative confocal images of FOXO3 (red) immunofluorescent staining in EL4 T cells infected with LV-Ctrl or LV-181a for 48 h through a ×63 oil immersion objective. Scale bars, 5 μm.(D and F) Luciferase activity analysis of reporter carrying Il23r promoter (D) or Ccl5 promoter (F) co-transfected into HEK293T with the indicated plasmids (n = 3–4 per group).(E) Real-time qRT-PCR analysis of Ccl5 expression in Th17 cells infected with FOXO3 lentivirus (LV-FOXO3) or control lentivirus (LV-control) (n = 4 per group). Data are presented as mean ± SEM of at least three independent experiments. In the statistical analysis, Student’s t test (A and B), One-way ANOVA (D, F), and Mann-Whitney U test (E) were used with significance defined as ∗p<0.05, ∗∗p<0.01. See also Figure S4.

Fig 5: The MYC/miR-181a-5p/AKT3 pathway in uveitis patients(A) Downregulated miR-181a-5p expression was shown in PBMCs from patients with BD (n = 6) (Puccetti et al., 2018) and enucleated globes of sympathetic ophthalmia (SO) (n = 4) (Kaneko et al., 2012). Reprinted by permission from Association for Research in Vision and Ophthalmology Publishers: Investigative Ophthalmology & Visual Science (Kaneko et al., 2012), Copyright 2012; and Hindnawi Publishers: Journal of Immunology Research (Puccetti et al., 2018), Copyright 2018.(B) Upregulated AKT3 expression was shown in PBMCs from BD patients by analysis of clinical dataset GSE70403(41 patients with BD and 17 normal volunteers).(C) Correlation between AKT3 gene expression and Th17 score in CD4+ T cells of BD patients was analyzed by the Pearson correlation test. The Th17 score was calculated based on the average expression level of IL-17, RORC, IL-23R, GM-CSF and CCL5 genes (n = 9).(D) Upregulated MYC expression was shown in CD4+ T cells of BD patients by analysis of clinical dataset GSE61399 (9 patients with BD and 3 healthy controls). The Z score transformed values of the microarray gene expression dataset GSE70403 (B) and GSE61399 (C, D). ∗p<0.05, ∗∗p<0.01.