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Gene expression profiling of A549 human lung cancer cell line treated with allosteric inhibitors targeting atypical PKCs

ABSTRACT: There is a current and ongoing need for improved cancer therapies. It is anticipated that future personalized cancer treatment will involve combinations of selective targeted drugs. A number of protein kinases, including members of the AGC group of kinases such as atypical protein kinase C (PKCi and PKCz) are validated drug targets. The regulation of many AGC protein kinases is mediated by a pocket in the small lobe of the kinase domain, the PIF-pocket. We developed a new compound class and provide biochemical and crystallography data showing that such compounds bind to the PIF-pocket and allosterically inhibit aPKC activity. We demonstrated that a representative compound, PS432, is a selective inhibitor in vivo. PS432, inhibited the proliferation of cancer cell lines more potently than ATM, a PKCi inhibitor in clinical trials, whereas PS432 had only minor effects on PNT1A cells. Analysis of the transcriptome by microarray and the proteome by SILAC indicated that the major effects of PS432 were on the cell cycle, while FACS indicated an accumulation of cells in the G1-G0 phase. PS432 significantly inhibited the growth of A549 tumor xenografts at 2.5 mg/kg/day without significant side effects. PS432 chemical class has good pharmacological properties and is orally available. Notably, PS432 had synergistic effects with a proteasome and with PI3-kinase inhibitors. The data indicates that derivatives from this new chemical class have potential for application in combination therapies in future personalized cancer treatments.

ORGANISM(S): Homo sapiens

PROVIDER: GSE63593 | GEO | 2017/01/17

SECONDARY ACCESSION(S): PRJNA268361

REPOSITORIES: GEO

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Project description:To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.

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Gene expression profiling of A549 human lung cancer cell line treated with allosteric inhibitors targeting atypical PKCs

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