A PDE6δ‐KRas Inhibitor Chemotype with up to Seven H‐Bonds and Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2

Martín‐Gago, Pablo; Fansa, Eyad K.; Klein, Christian; Murarka, Sandip; Janning, Petra; Schürmann, Marc; Metz, Malte; Ismail, Shehab; Schultz‐Fademrecht, Carsten; Baumann, Matthias; Bastiaens, Philippe I. H.; Wittinghofer, Alfred; Waldmann, Herbert

doi:10.1002/ange.201610957

Cited by 30 publications

(47 citation statements)

References 37 publications

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“…2a ). We also directly monitored the effect of Deltarasin on the interaction between Rheb and PDEδ by fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET), with mCitrine-Rheb as the donor and PDEδ, C-terminally tagged with mCherry (mCherry-PDEδ) as FRET acceptor (29, 32, 33) ( Fig. 2b ).…”

Section: Resultsmentioning

confidence: 99%

A spatially regulated GTPase cycle of Rheb controls growth factor signaling to mTORC1

Kovačević

Roßmannek

et al. 2018

Preprint

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19Growth factors initiate anabolism by activating mechanistic target of rapamycin complex 1 20 (mTORC1) via the small GTPase Rheb. We show that the GTPase cycle of Rheb is spatially 21 regulated by the interaction with its GDI-like solubilizing factor (GSF) -PDEδ. Arl2-GTP 22 mediated localized release of cytosolic Rheb-GTP from PDEδ deposits it onto perinuclear 23 membranes where it forms a complex with mTORC1. The membrane associated GTPase 24 activating protein (GAP) TSC2 hydrolyzes Rheb-GTP, weakening the interaction with mTOR. 25Rheb-GDP is readily released into the cytosol where it is maintained soluble by interaction 26 with PDEδ. This solubilized Rheb is re-activated by nucleotide exchange to be re-deposited 27 by Arl2-mediated release onto perinuclear membranes. This spatial GTPase cycle thereby 28 enables mTORC1 activation to be solely controlled by growth factor induced inactivation of 29 TSC2. The coupling between mTOR activation and spatially regulated Rheb nucleotide 30 exchange makes growth factor induced proliferation critically dependent on PDEδ 31 expression. 32 33 102 6 different time points after insulin stimulation of serum-starved TSC2+/+ MEFs also displayed 103 a transient recruitment of endogenous Rheb to mTOR containing membranes (Fig. 1d). 104 Enrichment of endogenous Rheb on mTOR-rich membranes was slightly delayed compared 105 to ectopically expressed mCitrine-Rheb, (30 min in the IF versus 12 min in the live-cell time 106 course), reflecting a shift in the reaction rate upon increasing the concentration of a reactant 107 according to the law of mass action. This transient increase in Rheb-enrichment on mTOR 108 containing membrane indicates that the interchange between the soluble and membrane-109 bound fraction of Rheb is dynamic, likely mediated and maintained by the solubilizing factor 110 PDEδ. 111 Arl2-mediated localized release from PDEδ deposits Rheb on perinuclear membranes 112 We examined how the interaction of Rheb with PDEδ influences its cytosol-membrane 113 partitioning by inhibiting PDEδ function using the small-molecule inhibitor Deltarasin that 114 targets PDEδ's prenyl-binding pocket (29). For this, we monitored the localization of Rheb, 115 N-terminally tagged with mCherry (mCherry-Rheb) as well as the hypervariable region of 116 Rheb, N-terminally tagged with mCitrine (mCitrine-Rheb HVR) in TSC2+/+ MEFs after 117 treatment with 3 µM Deltarasin. This resulted in a gradual loss of the perinuclear enrichment 118 for both proteins, demonstrating that the interaction of PDEδ via the farnesyl group in the 119 HVR is necessary for generating the perinuclear concentration of Rheb (Fig. 2a). We also 120 directly monitored the effect of Deltarasin on the interaction between Rheb and PDEδ by 121 fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET), 122 with mCitrine-Rheb as the donor and PDEδ, C-terminally tagged with mCherry (mCherry-PDEδ) as FRET acceptor (29, 32, 33) (Fig. 2b). The interaction between mCitrine-Rheb and 124 mCherry-PDEδ w...

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Section: Resultsmentioning

confidence: 99%

A spatially regulated GTPase cycle of Rheb controls growth factor signaling to mTORC1

Kovačević

Roßmannek

et al. 2018

Preprint

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“…Recent studies have solved structures of human farnesylated-methylated KRAS4b in complex with PDEδ in two different crystal forms. 30,31 Zimmermann and colleagues have reported that a small molecule inhibitor (deltarasin) binds to the prenyl binding pocket of PDEδ and thereby inhibits the KRAS-PDEδ interaction, which impairs KRAS signaling pathway and reduces the proliferation of human pancreatic ductal adenocarcinoma cells in vitro and in vivo. 32 However, subsequent detailed analysis of the characteristic doseresponse curves of deltarasin reveals that this PDEδ ligand displays a 'switch-like' inhibition of proliferation exhibiting cytotoxic effects at concentrations above 9 μM in all tested cell lines, including cancer and normal cells.…”

Section: Introductionmentioning

confidence: 99%

Identification of a new inhibitor of KRAS‐PDEδ interaction targeting KRAS mutant nonsmall cell lung cancer

Leung

Luo

Liu

et al. 2019

Intl Journal of Cancer

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Oncogenic KRAS is considered a promising target for anti‐cancer therapy. However, direct pharmacological strategies targeting KRAS‐driven cancers remained unavailable. The prenyl‐binding protein PDEδ, a transporter of KRAS, has been identified as a potential target for pharmacological inhibitor by selectively binding to its prenyl‐binding pocket, impairing oncogenic KRAS signaling pathway. Here, we discovered a novel PDEδ inhibitor (E)‐N′‐((3‐(tert‐butyl)‐2‐hydroxy‐6,7,8,9‐tetrahydrodibenzo[b,dfuran‐1‐yl)methylene)‐2,4‐dihydroxybenzohydrazide(NHTD) by using a high‐throughput docking‐based virtual screening approach. In vitro and in vivo studies demonstrated that NHTD suppressed proliferation, induced apoptosis and inhibited oncogenic K‐RAS signaling pathways by disrupting KRAS‐PDEδ interaction in nonsmall cell lung cancer (NSCLC) harboring KRAS mutations. NHTD redistributed the localization of KRAS to endomembranes by targeting the prenyl‐binding pocket of PDEδ and exhibited the suppression of abnormal KRAS function. Importantly, NHTD prevented tumor growth in xenograft and KRAS mutant mouse model, which presents an effective strategy targeting KRAS‐driven cancer.

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“…Small-molecule inhibitors that potently target the PDEd prenyl-binding site, for example, deltasonamide 1 (Figure 1), have proven invaluable for the study of PDEd function. [6][7][8][9] However, their application is limited by an allosteric interaction of PDEd with the Arl2/3 GTPases, which results in release of even high-affinity cargo. [4,8,9] An alternative approach to inhibition consists of eventdriven pharmacology, for example, small-molecule-induced protein degradation.…”

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confidence: 99%

“…[6][7][8][9] However, their application is limited by an allosteric interaction of PDEd with the Arl2/3 GTPases, which results in release of even high-affinity cargo. [4,8,9] An alternative approach to inhibition consists of eventdriven pharmacology, for example, small-molecule-induced protein degradation. For this purpose, heterobifunctional molecules are employed that bind the protein of interest and recruit an E3 ubiquitin ligase, followed by ubiquitination and subsequent degradation of the targeted protein.…”

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confidence: 99%

“…However, the benzyl derivative, which displays 5-fold lowered affinity, offers a simplified synthesis for the attachment of a linker ( Figure 1). [8] Moreover, PROTAC-mediated degradation is event-driven and the low nanomolar affinity of the benzyl analogue for PDEd would most likely be sufficient to trigger target degradation. Thus, we designed PROTACs based on the benzyl derivative of deltasonamide 1, where the E3 ligase ligand was attached via an oligoethylene glycol linker to the benzoic acid ( Figure 1).…”

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Development of a PDEδ‐Targeting PROTACs that Impair Lipid Metabolism

et al. 2020

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The prenyl‐protein chaperone PDEδ modulates the localization of lipidated proteins in the cell, but current knowledge about its biological function is limited. Small‐molecule inhibitors that target the PDEδ prenyl‐binding site have proven invaluable in the analysis of biological processes mediated by PDEδ, like KRas cellular trafficking. However, allosteric inhibitor release from PDEδ by the Arl2/3 GTPases limits their application. We describe the development of new proteolysis‐targeting chimeras (PROTACs) that efficiently and selectively reduce PDEδ levels in cells through induced proteasomal degradation. Application of the PDEδ PROTACs increased sterol regulatory element binding protein (SREBP)‐mediated gene expression of enzymes involved in lipid metabolism, which was accompanied by elevated levels of cholesterol precursors. This finding for the first time demonstrates that PDEδ function plays a role in the regulation of enzymes of the mevalonate pathway.

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A PDE6δ‐KRas Inhibitor Chemotype with up to Seven H‐Bonds and Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2

Cited by 30 publications

References 37 publications

A spatially regulated GTPase cycle of Rheb controls growth factor signaling to mTORC1

A spatially regulated GTPase cycle of Rheb controls growth factor signaling to mTORC1

Identification of a new inhibitor of KRAS‐PDEδ interaction targeting KRAS mutant nonsmall cell lung cancer

Development of a PDEδ‐Targeting PROTACs that Impair Lipid Metabolism

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