ATP-competitive and allosteric inhibitors induce differential conformational changes at the autoinhibitory interface of Akt
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Abstract
The protein kinase Akt is a master regulator of pro-growth signalling in the cell. Akt is activated through its targeted recruitment to phosphoinositides, leading to disruption of the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains. Hyper activation of Akt is common in oncogenic transformation, with multiple oncogenic activating mutants identified in Akt. This has led to the development of potent and selective ATP-competitive and allosteric inhibitors for Akt. Paradoxically, some ATP-competitive Akt inhibitors cause hyperphosphorylation of Akt. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding to the Akt active site inhibitor A-443654, and the Akt allosteric inhibitor MK-2206. We compared the conformational changes that occurred for each inhibitor under three different states of Akt: i- inactive monophosphorylated, ii- partially active tris-phosphorylated [T308, T450, S473], and iii- fully activated, tris-phosphorylated bound to PIP 3 membranes. The allosteric MK-2206 inhibitor results in large scale allosteric conformational changes in all states, and restricts membrane binding through sequestration of the PH domain. Binding of the A-443654 inhibitor led to large scale allosteric conformational changes in both the monophosphorylated and phosphorylated states, leading to an alteration in the autoinhibitory PH-kinase interface. We also observed increased protection in the PH domain upon membrane binding in the presence of A-443654, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and dynamic conformational changes induced by Akt inhibitors, and has important implications for the design of Akt targeted therapeutics.
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Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Ruchika Bajaj and Sanjay Kumar Sukumar. Review synthesized by Iratxe Puebla.
The preprint studied the conformational changes upon binding of the Akt protein kinase to the Akt active site inhibitor A-443654 and the Akt allosteric inhibitor MK-2206, under three states of Akt: inactive monophosphorylated, partially active tris-phosphorylated, and fully activated, tris-phosphorylated bound to PIP3 membranes. The MK-2206 resulted in allosteric conformational changes in all states and restricted membrane binding through sequestration of the PH domain. The A-443654 inhibitor led to allosteric conformational changes in the monophosphorylated and phosphorylated states, with increased protection in the PH domain upon membrane binding. The …
Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Ruchika Bajaj and Sanjay Kumar Sukumar. Review synthesized by Iratxe Puebla.
The preprint studied the conformational changes upon binding of the Akt protein kinase to the Akt active site inhibitor A-443654 and the Akt allosteric inhibitor MK-2206, under three states of Akt: inactive monophosphorylated, partially active tris-phosphorylated, and fully activated, tris-phosphorylated bound to PIP3 membranes. The MK-2206 resulted in allosteric conformational changes in all states and restricted membrane binding through sequestration of the PH domain. The A-443654 inhibitor led to allosteric conformational changes in the monophosphorylated and phosphorylated states, with increased protection in the PH domain upon membrane binding. The results can assist the design of Akt-targeted therapeutics.
The reviewers had a few minor comments about the paper:
- It could be helpful to include a short explanation early in the text about the use of HDX-MS, how it works and why it is useful for exploring conformational changes.
- Figure 2A+B provide a nice representation of the HDX exchange data.
- Results ‘3 seconds at 1°C, which is referred to as 0.3 sec in all graphs and the source data)’ - This may be a bit confusing for someone who wants to look at the data in the figures independently. Consider an alternative way of representation or providing some further clarification in the figure legend.
- Results ‘Decreases in exchange in the kinase domain were similar to those observed in the absence of membranes, occurring in regions encompassing the αC helix, the ATP binding pocket, as well as changes covering the activation loop and C-lobe:PH interface’ - Please clarify whether the comparison here relates to the data in Fig. 3A/C vs Fig 4A/C.
- Results ‘There were multiple regions of significantly decreased deuterium exchange in the kinase and PH domains (Fig. 2B, 2D, 2E).’ - This section mainly focuses on conformational change upon the addition of MK-2206 allosteric inhibitor binding. Figure 2F appears to be the most relevant for the comparison. It is suggested to provide additional combination of data with ATP analogs to understand the coordination of ATP and inhibitor during the inhibition step in the cycle.
- Results ‘Both experiments were carried out under saturating concentrations of inhibitor binding, so this difference reflects intrinsic conformational differences.’ - Saturating concentrations implies that most of the population will be in the same conformation. Please comment on the association between saturating concentrations and intrinsic conformational differences.
- Discussion - There do not appear to be many structures available for different conformational states of Akt. The study has mapped hdx data on available structures, however, it'd be good to see correlation of conformational changes by HDX with conformational changes in structure.
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