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: Generating Ba/F3 myr-Akt isoform-dependent cell lines to evaluate targeted Akt inhibition.a Representation of the IL-3 signaling pathway in the murine Ba/F3 cell upon binding of IL-3. b General overview of the established cell lines Ba/F3myr-Akt1 (gray), Ba/F3myr-Akt2 (red), and Ba/F3myr-Akt3 (yellow). Graph showing the seeded cell number of the Ba/F3 constructs and resulting luminescent signals following treatment with CTG solution compared to the parental system (dotted line, white). The experiments were performed in triplicates with n = 3. Error bars indicate s.d. Immunoblot of myr-Akt isoform systems highlighting the molecular expression levels, activities, and alterations of downstream targets of Akt (n = 1 biologically independent experiment). c Addressing the Ba/F3 myr-Akt with CAAI borussertib to investigate the sensitivity of the established system. Structure of used inhibitor and schematic representation of the binding mode. Graph showing the remaining viable cells as a percentage with respect to the used concentration of borussertib. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Immunoblots resolved individual Akt isoforms’ presence and activity within the three model systems after 4-h treatment (n = 1 biologically independent experiment). d Addressing the Ba/F3 myr-Akt cells with isoform-selective CAAIs 15a/16b to investigate selectivity translation into the established system. Graph showing the remaining viable cells as a percentage with respect to the used concentration of 15a and 16b. Experimental points were measured in duplicates for each plate and were replicated in n = 3 biologically independent experiments. Structure of used inhibitors. Immunoblots resolved isoform selective activity within the three model systems after 4-h treatment (n = 1 biologically independent experiment). In c, d the box plot middle line represents the mean value, the box represents the first and third quartile and the error bars indicate s.e. Source Data are provided with this paper.

Fig 4: AKT1 Phosphorylates HSF1 on S326 and induces HSF1 activity independent of mTORC1. (A-C) Indicated cell lines were transfected with vector, AKT1, AKT2 or AKT3 along with the HSE luciferase reporter for 48 h. Firefly/Renilla ratios were compared across groups. (D) Recombinant proteins were incubated at 30 °C for 2 h in the presence of ATP and subsequently subjected to SDS/PAGE and immunoblotting with indicated antibodies. (E) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cell lysates were subjected to SDS/PAGE and immunoblotting with indicated antibodies. (F-G) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 in the presence or absence of rapamycin (10–20 nm). Cell lysates were subjected to firefly reporter assay (F) or immunoblotting with indicated antibodies (G). Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. *P < 0.05.

Fig 5: 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.