ABSTRACT
The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.
Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays , 1-Phosphatidylinositol 4-Kinase/chemistry , 1-Phosphatidylinositol 4-Kinase/metabolism , Animals , Biological Transport , COS Cells , Chlorocebus aethiops , Drug Evaluation, Preclinical , Endosomes/drug effects , Endosomes/metabolism , Enzyme Inhibitors/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , HEK293 Cells , Humans , Molecular Docking Simulation , Protein ConformationABSTRACT
Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X-ray crystallography.
Subject(s)
Bacteriophage T4 , Chromatography, Affinity/methods , Muramidase , Mutation , Bacteriophage T4/enzymology , Bacteriophage T4/genetics , Crystallography, X-Ray/methods , Muramidase/chemistry , Muramidase/genetics , Muramidase/isolation & purificationABSTRACT
Picornaviruses are small positive-sense single-stranded RNA viruses that include many important human pathogens. Within the host cell, they replicate at specific replication sites called replication organelles. To create this membrane platform, they hijack several host factors including the acyl-CoA-binding domain-containing protein-3 (ACBD3). Here, we present a structural characterization of the molecular complexes formed by the non-structural 3A proteins from two species of the Kobuvirus genus of the Picornaviridae family and the 3A-binding domain of the host ACBD3 protein. Specifically, we present a series of crystal structures as well as a molecular dynamics simulation of the 3A:ACBD3 complex at the membrane, which reveals that the viral 3A proteins act as molecular harnesses to enslave the ACBD3 protein leading to its stabilization at target membranes. Our data provide a structural rationale for understanding how these viral-host protein complexes assemble at the atomic level and identify new potential targets for antiviral therapies.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Host-Pathogen Interactions , Kobuvirus/genetics , Membrane Proteins/chemistry , Unilamellar Liposomes/chemistry , Viral Nonstructural Proteins/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs , Binding Sites , Cell Line , Cloning, Molecular , Crystallography, X-Ray , Gene Expression , Humans , Kobuvirus/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/geneticsABSTRACT
Phosphatidylinositol 4-kinase IIIß (PI4KB) is indispensable for the replication of various positive-sense single stranded RNA viruses, which hijack this cellular enzyme to remodel intracellular membranes of infected cells to set up the functional replication machinery. Therefore, the inhibition of this PI4K isoform leads to the arrest of viral replication. Here, we report on the synthesis of novel PI4KB inhibitors, which were rationally designed based on two distinct structural types of inhibitors that bind in the ATP binding side of PI4KB. These "hybrids" not only excel in outstanding inhibitory activity but also show high selectivity to PI4KB compared to other kinases. Thus, these compounds exert selective nanomolar or even subnanomolar activity against PI4KB as well as profound antiviral effect against hepatitis C virus, human rhinovirus, and coxsackievirus B3. Our crystallographic analysis unveiled the exact position of the side chains and explains their extensive contribution to the inhibitory activity.
Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Drug Design , HeLa Cells , Humans , Molecular StructureABSTRACT
Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , Binding Sites , COS Cells , Chlorocebus aethiops , Golgi Apparatus/metabolism , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Phosphatidylinositol Phosphates/metabolism , Protein Binding , Protein Structure, SecondaryABSTRACT
Phosphatidylinositol 4-phosphate (PI4P) is the most abundant monophosphoinositide in eukaryotic cells. Humans have four phosphatidylinositol 4-kinases (PI4Ks) that synthesize PI4P, among which are PI4K IIß and PI4K IIα. In this study, two crystal structures are presented: the structure of human PI4K IIß and the structure of PI4K IIα containing a nucleoside analogue. The former, a complex with ATP, is the first high-resolution (1.9â Å) structure of a PI4K. These structures reveal new details such as high conformational heterogeneity of the lateral hydrophobic pocket of the C-lobe and together provide a structural basis for isoform-specific inhibitor design.
Subject(s)
1-Phosphatidylinositol 4-Kinase/chemistry , Drug Design , Nucleosides/chemistry , Protein Kinase Inhibitors/chemistry , 1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , 1-Phosphatidylinositol 4-Kinase/metabolism , Adenosine Triphosphate/metabolism , Catalytic Domain , Crystallography, X-Ray , Humans , Hydrophobic and Hydrophilic Interactions , Nucleosides/pharmacology , Protein Conformation , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Kinase Inhibitors/pharmacologyABSTRACT
Phosphatidylinositol 4-kinase IIIß is a cellular lipid kinase pivotal to pathogenesis of various RNA viruses. These viruses hijack the enzyme in order to modify the structure of intracellular membranes and use them for the construction of functional replication machinery. Selective inhibitors of this enzyme are potential broad-spectrum antiviral agents, as inhibition of this enzyme results in the arrest of replication of PI4K IIIß-dependent viruses. Herein, we report a detailed study of novel selective inhibitors of PI4K IIIß, which exert antiviral activity against a panel of single-stranded positive-sense RNA viruses. Our crystallographic data show that the inhibitors occupy the binding site for the adenine ring of the ATP molecule and therefore prevent the phosphorylation reaction.
Subject(s)
Antiviral Agents/chemistry , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Binding Sites , Crystallography, X-Ray , Ethylenediamines/chemical synthesis , Ethylenediamines/chemistry , Ethylenediamines/pharmacology , HeLa Cells , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Imidazoles/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Molecular Docking Simulation , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Protein Conformation , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyridines/chemical synthesis , Pyridines/chemistry , Pyridines/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Pyrimidines/pharmacology , RNA Viruses/drug effects , Structure-Activity Relationship , Triazines/chemical synthesis , Triazines/chemistry , Triazines/pharmacologyABSTRACT
Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα), in complex with ATP solved by X-ray crystallography at 2.8 Å resolution. The structure revealed a non-typical kinase fold that could be divided into N- and C-lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C-lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.
