KRas4B-PDE6δ complex stabilization by small molecules obtained by virtual screening affects Ras signaling in pancreatic cancer

Abstract

BackgroundThe GTPase KRas4B has been utilized as a principal target in the development of anticancer drugs. PDE6δ transports KRas4B to the plasma membrane, where it is released to activate various signaling pathways required for the initiation and maintenance of cancer. Therefore, identifying new small molecules that prevent activation of this GTPase by stabilizing the KRas4B-PDE6δ molecular complex is a practical strategy to fight against cancer.MethodsThe crystal structure of the KRas4B-PDE6δ heterodimer was employed to locate possible specific binding sites at the protein-protein interface region. Virtual screening of Enamine-database compounds was performed on the located potential binding sites to identify ligands able to simultaneously bind to the KRas4B-PDE6δ heterodimer. A molecular dynamics approach was used to estimate the binding free-energy of the complex. Cell viability and apoptosis were measured by flow cytometry. G-LISA was used to measure Ras inactivation. Western blot was used to measure AKT and ERK activation. MIA PaCa-2 cells implanted subcutaneously into nude mice were treated with D14 or C22 and tumor volumes were recorded.ResultsAccording to the binding affinity estimation, D14 and C22 stabilized the protein-protein interaction in the KRas4B-PDE6δ complex based on in vitro evaluation of the 38 compounds showing antineoplastic activity against pancreatic MIA PaCa-2 cancer cells. In this work, we further investigated the antineoplastic cellular properties of two of them, termed D14 and C22, which reduced the viability in the human pancreatic cancer cells lines MIA PaCa-2, PanC-1 and BxPC-3, but not in the normal pancreatic cell line hTERT-HPNE. Compounds D14 and C22 induced cellular death via apoptosis. D14 and C22 significantly decreased Ras-GTP activity by 33% in MIA PaCa-2 cells. Moreover, D14 decreased AKT phosphorylation by 70% and ERK phosphorylation by 51%, while compound C22 reduced AKT phosphorylation by 60% and ERK phosphorylation by 36%. In addition, compounds C22 and D14 significantly reduced tumor growth by 88.6 and 65.9%, respectively, in a mouse xenograft model.ConclusionsWe identified two promising compounds, D14 and C22, that might be useful as therapeutic drugs for pancreatic ductal adenocarcinoma treatment.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-5142-7) contains supplementary material, which is available to authorized users.

Molecular mechanism of the association and dissociation of Deltarasin from the heterodimeric KRas4B‐PDEδ complex

“…This result indicates that Deltarasin can exert two inhibitory mechanisms for KRas4B‐PDEδ‐HVR2 complex. When G12C, G13D, Q61L, and Q61R mutations are present, Deltarasin contributes to stabilize the KRas4B‐PDEδ‐HVR2 complex, preventing the release of KRas4B from the plasma membrane to inhibit Ras signaling, as previously observed for some specific inhibitors of KRas4B signaling in pancreatic and colon cancer . Whereas that in WT and when G12D and G12V mutations are present, Deltarasin exerts an inhibitory activity by promoting the dissociation of the KRas4B‐PDEδ‐HVR2, through a similar mechanism than that observed for Deltarasin blocking PDEδ and subsequently preventing the formation of the KRas4B‐PDEδ‐HVR2 …”

“…Although Deltarasin was designed before the crystal structure of the KRas4B‐PDEδ complex was solved, information about the structure of the KRas4B‐PDEδ complex was recently released, revealing the presence of a second conformations of the KRas4B‐PDEδ complex which have been employed to explore the ability of ligands to bind at the protein‐protein interface of KRas4B‐PDEδ . More recently, specific inhibitors of KRas4B signaling in pancreatic and colon cancer have been developed by our research group . In contrast to Deltarasin that inhibits the formation of the KRas4B‐PDEδ complex by binding to PDEδ, these inhibitors stabilize the KRas4B‐PDEδ complex by increasing the protein‐protein affinity and subsequently preventing the release of KRas4B from the plasma membrane to inhibit Ras signaling .…”

“…More recently, specific inhibitors of KRas4B signaling in pancreatic and colon cancer have been developed by our research group . In contrast to Deltarasin that inhibits the formation of the KRas4B‐PDEδ complex by binding to PDEδ, these inhibitors stabilize the KRas4B‐PDEδ complex by increasing the protein‐protein affinity and subsequently preventing the release of KRas4B from the plasma membrane to inhibit Ras signaling . Thus, Deltarasin may also alter the affinity of the KRas4B‐PDEδ complex by binding to the protein‐protein interface.…”

KRas4BG12C/D/PDE6δ Heterodimeric Molecular Complex: A Target Molecular Multicomplex for the Identification and Evaluation of Nontoxic Pharmacological Compounds for the Treatment of Pancreatic Cancer

“…This compound was named the first generation of PDE6δ inhibitors [26]. However, its evaluation in noncancerous pancreatic duct cell lines showed high cytotoxicity; this affected considerably the cell viability at low concentrations [28]. In 2016, the analogue of the compound Deltarasin (second generation of PDE6δ inhibitors) was reported, and it was called deltazinone.…”

“…The identification of the compounds that had an in silico interaction with the complex, in addition these compounds was selected considering that they complied with the Lipinsky rule, which states that (1) the compounds should not have more than five hydrogen bridge donors; (2) they must not contain more than 10 hydrogen bridge acceptors; (3) they must have a molecular weight of less than 500 g/mol; (4) the compounds must have an octanol/water partition coefficient of less than five (log P < 5). Compounds identified as D14 and C22 showed different in silico interaction energies on the KRas4B/PDE6δ and K-Ras4BG12C/PDE6δ heterocomplex crystals; these interaction energies ranged from −143 to −162 ΔG [28].…”