ABSTRACT
The acyl-binding UNC119 proteins mediate the activation and transport of various N-myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α-helical peptides that target UNC119a. By using the hydrocarbon peptide-stapling approach, cell-permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Lipid Metabolism , Peptides/metabolism , Peptides/pharmacology , Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Sequence , HeLa Cells , Humans , Models, Molecular , Molecular Targeted Therapy , Peptides/chemistry , Protein Binding , Protein Conformation, alpha-HelicalABSTRACT
Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Available inhibitors target Smoothened (Smo), which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smo are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility in vitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis by inducing spindle misorientation and pseudoprometaphase delay. Dynarrestin reversibly inhibits cytoplasmic dynein 2-dependent intraflagellar transport (IFT) of the cargo IFT88 and flux of Smo within cilia without interfering with ciliogenesis and suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. As such, dynarrestin is a valuable tool for probing cytoplasmic dynein-dependent cellular processes and a promising compound for medicinal chemistry programs aimed at development of anti-cancer drugs.
Subject(s)
Cytoplasmic Dyneins/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Animals , Biological Transport/drug effects , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cilia/drug effects , Cilia/metabolism , Cytoplasmic Dyneins/metabolism , Hedgehog Proteins/antagonists & inhibitors , Hedgehog Proteins/metabolism , Humans , Mice , Mitosis/drug effects , NIH 3T3 Cells , Protein Transport/drug effects , Signal Transduction/drug effectsABSTRACT
Prenylation is a post-translational modification that increases the affinity of proteins for membranes and mediates protein-protein interactions. The retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ) is a prenyl binding protein that is essential for the shuttling of small GTPases between different membrane compartments and, thus, for their proper functioning. Although the prenylome comprises up to 2% of the mammalian proteome, only few prenylated proteins are known to interact with PDEδ. A proteome-wide approach was employed to map the PDEδ interactome among the prenylome and revealed RAB23, CDC42 and CNP as novel PDEδ interacting proteins. Moreover, PDEδ associates with the lamin A mutant progerin in a prenyl-dependent manner. These findings shed new light on the role of PDEδ in binding (and regulating) prenylated proteins in cells.
Subject(s)
Carrier Proteins/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Carrier Proteins/chemistry , Cells, Cultured , Cyclic Nucleotide Phosphodiesterases, Type 6/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Structure , Protein Binding , Structure-Activity RelationshipABSTRACT
Small GTPases comprise a family of highly relevant targets in chemical biology and medicinal chemistry research and have been considered "undruggable" due to the persisting lack of effective synthetic modulators and suitable binding pockets. As molecular switches, small GTPases control a multitude of pivotal cellular functions, and their dysregulation is associated with many human diseases such as various forms of cancer. Rab-GTPases represent the largest subfamily of small GTPases and are master regulators of vesicular transport interacting with various proteins via flat and extensive protein-protein interactions (PPIs). The only reported synthetic inhibitor of a PPI involving an activated Rab GTPase is the hydrocarbon stapled peptide StRIP3. However, this macrocyclic peptide shows low proteolytic stability and cell permeability. Here, we report the design of a bioavailable StRIP3 analogue that harbors two hydrophobic cross-links and exhibits increased binding affinity, combined with robust cellular uptake and extremely high proteolytic stability. Localization experiments reveal that this double-stapled peptide and its target protein Rab8a accumulate in the same cellular compartments. The reported approach offers a strategy for the implementation of biostability into conformationally constrained peptides while supporting cellular uptake and target affinity, thereby conveying drug-like properties.
Subject(s)
Peptide Hydrolases/metabolism , Peptides/metabolism , rab GTP-Binding Proteins/metabolism , Amino Acid Sequence , Biological Availability , HeLa Cells , Humans , Peptides/chemistry , PermeabilityABSTRACT
Holoprosencephaly is a common developmental disorder in humans characterised by incomplete brain hemisphere separation and midface anomalies. The etiology of holoprosencephaly is heterogeneous with environmental and genetic causes, but for a majority of holoprosencephaly cases the genes associated with the pathogenesis could not be identified so far. Here we report the generation of knockout mice for the ubiquitin E3 ligase NOSIP. The loss of NOSIP in mice causes holoprosencephaly and facial anomalies including cleft lip/palate, cyclopia and facial midline clefting. By a mass spectrometry based protein interaction screen we identified NOSIP as a novel interaction partner of protein phosphatase PP2A. NOSIP mediates the monoubiquitination of the PP2A catalytic subunit and the loss of NOSIP results in an increase in PP2A activity in craniofacial tissue in NOSIP knockout mice. We conclude, that NOSIP is a critical modulator of brain and craniofacial development in mice and a candidate gene for holoprosencephaly in humans.
Subject(s)
Face/embryology , Protein Phosphatase 2/metabolism , Skull/embryology , Skull/enzymology , Ubiquitin-Protein Ligases/metabolism , Animals , Animals, Newborn , Catalytic Domain , Cleft Palate/embryology , Cleft Palate/enzymology , Face/abnormalities , Holoprosencephaly/embryology , Holoprosencephaly/enzymology , Holoprosencephaly/pathology , Methylation , Mice, Inbred C57BL , Mice, Knockout , Protein Binding , Skull/abnormalities , UbiquitinationABSTRACT
K-Ras is one of the most frequently mutated signal transducing human oncogenes. Ras signaling activity requires correct cellular localization of the GTPase. The spatial organization of K-Ras is controlled by the prenyl binding protein PDEδ, which enhances Ras diffusion in the cytosol. Inhibition of the Ras-PDEδ interaction by small molecules impairs Ras localization and signaling. Here we describe in detail the identification and structure guided development of Ras-PDEδ inhibitors targeting the farnesyl binding pocket of PDEδ with nanomolar affinity. We report kinetic data that characterize the binding of the most potent small molecule ligands to PDEδ and prove their binding to endogenous PDEδ in cell lysates. The PDEδ inhibitors provide promising starting points for the establishment of new drug discovery programs aimed at cancers harboring oncogenic K-Ras.
Subject(s)
Benzimidazoles/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 6/antagonists & inhibitors , Atorvastatin , Benzimidazoles/chemical synthesis , Benzimidazoles/pharmacology , Binding Sites , Cell Line, Tumor , Computer Simulation , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Drug Design , Esters , Ethers/chemical synthesis , Ethers/chemistry , Ethers/pharmacology , Heptanoic Acids/chemical synthesis , Heptanoic Acids/chemistry , Heptanoic Acids/pharmacology , Humans , Kinetics , Models, Molecular , Protein Prenylation , Pyrroles/chemical synthesis , Pyrroles/chemistry , Pyrroles/pharmacology , Stereoisomerism , Structure-Activity Relationship , Thermodynamics , ras Proteins/metabolismABSTRACT
The imino Diels-Alder reaction is an efficient method for the synthesis of aza-heterocycles. While different stereo- and enantioselective inverse-electron-demand imino Diels-Alder (IEDIDA) reactions have been reported before, IEDIDA reactions including electron-deficient dienes are unprecedented. The first enantioselective IEDIDA reaction between electron-poor chromone dienes and cyclic imines, catalyzed by zinc/binol complexes is described. The novel reaction provides a facile entry to a natural product inspired collection of ring-fused quinolizines including a potent modulator of mitosis.