ABSTRACT
The Src-family kinase Fgr is expressed primarily in myeloid hematopoietic cells and contributes to myeloid leukemia. Here, we present X-ray crystal structures of Fgr bound to the ATP-site inhibitors A-419259 and TL02-59, which show promise as anti-leukemic agents. A-419259 induces a closed Fgr conformation, with the SH3 and SH2 domains engaging the SH2-kinase linker and C-terminal tail, respectively. In the Fgr:A-419259 complex, the activation loop of one monomer inserts into the active site of the other, providing a snapshot of trans-autophosphorylation. By contrast, TL02-59 binding induced SH2 domain displacement from the C-terminal tail and SH3 domain release from the linker. Solution studies using HDX MS were consistent with the crystal structures, with A-419259 reducing and TL02-59 enhancing solvent exposure of the SH3 domain. These structures demonstrate that allosteric connections between the kinase and regulatory domains of Src-family kinases are regulated by the ligand bound to the active site.
Subject(s)
Leukemia, Myeloid, Acute , src-Family Kinases , Humans , src-Family Kinases/chemistry , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/metabolism , src Homology Domains , Leukemia, Myeloid, Acute/drug therapy , Adenosine TriphosphateABSTRACT
Ponatinib is a multi-target tyrosine kinase inhibitor that targets ABL, SRC, FGFR, and so on. It was designed to overcome the resistance of BCR-ABL mutation to imatinib, especially the gatekeeper mutation ABLT315I. The molecular mechanism by which ponatinib overcomes mutations of BCR-ABL and some other targets has been explained, but little information is known about the characteristics of ponatinib binding to SRC. Here, we showed that ponatinib inhibited wild type SRC kinase but failed to inhibit SRC gatekeeper mutants in both biochemical and cellular assays. We determined the crystal structure of ponatinib in complex with the SRC kinase domain. In addition, by structural analysis, we provided a possible explanation for why ponatinib showed different effects on SRC and other kinases with gatekeeper mutations. The resistance mechanism of SRC gatekeeper mutations to ponatinib may provide meaningful information for designing inhibitors against SRC family kinases in the future.
Subject(s)
Imidazoles/chemistry , Imidazoles/pharmacology , Protein Kinase Inhibitors/chemistry , Pyridazines/chemistry , Pyridazines/pharmacology , src-Family Kinases/chemistry , Binding Sites , Crystallography, X-Ray , Humans , Imidazoles/metabolism , Models, Molecular , Mutation , Protein Conformation , Protein Domains , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/metabolism , Pyridazines/metabolism , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID-19 patients. Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases. Curcumin has been previously identified as an excellent DYRK-2 inhibitor, but curcumin's fate is tainted by its instability in the cellular environment. Besides, small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity. Here, we show that functionalization of the 4-arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor (Kd = 50 ± 10 nM). The mustard curcumin derivative preferentially interacts with the inactive conformation of Hck, similar to type-II kinase inhibitors that are less promiscuous. Moreover, the lead compound showed no inhibitory effect on three other kinases (DYRK2, Src, and Abl). We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signaling pathway in triple-negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodeling its target affinity and cellular stability.
Subject(s)
Curcumin/pharmacology , Drug Design , Epithelial Cells/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Animals , Cell Line, Tumor , Cloning, Molecular , Curcumin/analogs & derivatives , Curcumin/chemical synthesis , Drug Stability , Epithelial Cells/enzymology , Epithelial Cells/pathology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , HT29 Cells , Humans , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/genetics , Proto-Oncogene Proteins c-hck/metabolism , RAW 264.7 Cells , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , src-Family Kinases/genetics , src-Family Kinases/metabolism , Dyrk KinasesABSTRACT
Protein-protein interactions are key to define the function of nucleotide binding domain (NBD) and leucine-rich repeat (LRR) family, pyrin domain (PYD)-containing protein 12 (NLRP12). cDNA encoding the human PYD + NBD of NLRP12 was used as bait in a yeast two-hybrid screen with a human leukocyte cDNA library as prey. Hematopoiesis cell kinase (HCK), a member of the c-SRC family of non-receptor tyrosine kinases, was among the top hits. The C-terminal 40 amino acids of HCK selectively bound to NLRP12's PYD + NBD, but not to that of NLRP3 and NLRP8. Amino acids F503, I506, Q507, L510, and D511 of HCK are critical for the binding of HCK's C-terminal 40 amino acids to NLRP12's PYD + NBD. Additionally, the C-terminal 30 amino acids of HCK are sufficient to bind to NLRP12's PYD + NBD, but not to its PYD alone nor to its NBD alone. In cell lines that express HCK endogenously, it was co- immunoprecipitated with stably expressed exogenous NLRP12. Also, NLRP12 co-immunoprecipitated and co-localized with HCK when both were overexpressed in 293T cells. In addition, in this overexpression system, steady-state NLRP12 protein expression levels significantly decreased when HCK was co-expressed. Bioinformatic analysis showed that HCK mRNA co-occurred with NLRP12 mRNA, but not with other NLRP mRNAs, in blood and marrow samples from acute myeloid leukemia (AML) patients. The mRNA of NLRP12 is also co-expressed with HCK in AML patient samples, and the levels of mRNA expression of each are correlated. Together these data suggest that NLRP12, through its binding to HCK, may have an effect on the pathogenesis of AML.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Proto-Oncogene Proteins c-hck/metabolism , Bone Marrow/metabolism , Cell Line , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/blood , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Leukemia, Myeloid, Acute/blood , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins c-hck/blood , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/genetics , Two-Hybrid System TechniquesABSTRACT
Src family kinases (SFKs) are a group of nonreceptor tyrosine kinases that are characterized by their involvement in critical signal transduction pathways. SFKs are often found attached to membranes, but little is known about the conformation of the protein in this environment. Here, solution nuclear magnetic resonance (NMR), neutron reflectometry (NR), and molecular dynamics (MD) simulations were employed to study the membrane interactions of the intrinsically disordered SH4 and Unique domains of the Src family kinase Hck. Through development of a procedure to combine the information from the different techniques, we were able produce a first-of-its-kind atomically detailed structural ensemble of a membrane-bound intrinsically disordered protein. Evaluation of the model demonstrated its consistency with previous work and provided insight into how SFK Unique domains act to differentiate the family members from one another. Fortuitously, the position of the ensemble on the membrane allowed the model to be combined with configurations of the multidomain Hck kinase previously determined from small-angle solution X-ray scattering to produce full-length models of membrane-anchored Hck. The resulting models allowed us to estimate that the kinase active site is positioned about 65 ± 35 Å away from the membrane surface, offering the first estimations of the length scale associated with the concept of SFK subcellular localization.
Subject(s)
Cell Membrane/metabolism , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/metabolism , Binding Sites , Catalytic Domain , Humans , Models, Molecular , Molecular Dynamics Simulation , Neutron Diffraction , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Domains , Scattering, Small AngleABSTRACT
Hematopoietic cell kinase (Hck) is an important signaling enzyme and a potential drug target for HIV infections and Bcr/Abl-chronic myeloid leukemia. The protein shares the same SH4-Unique-SH3-SH2-kinase multi-domain architecture as the other eight members of the Src family of non-receptor tyrosine kinases. These enzymes are often found anchored to the intracellular side of the membrane via lipidation of the SH4 domain and are integral components of signaling cascades localized at the cell surface. Despite the detailed structural information available for the SH3, SH2, and kinase domains of Hck, the intrinsically disordered nature of the SH4 and Unique domains has resulted in a lack of information for this important region of the protein that is responsible for membrane association. Here, we report the 1H, 15N and 13C chemical shifts of the Hck SH4-Unique domains at pH 4.5.
Subject(s)
Intrinsically Disordered Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular , Proto-Oncogene Proteins c-hck/chemistry , Carbon Isotopes , Humans , Nitrogen Isotopes , Protein Domains , ProtonsABSTRACT
A series of rhodanine derivatives was synthesized in the Knoevenagel condensation of rhodanine and different aldehydes using choline chloride:urea (1:2) deep eutectic solvent. This environmentally friendly and catalyst free approach was very effective in the condensation of rhodanine with commercially available aldehydes, as well as the ones synthesized in our laboratory. All rhodanine derivatives were subjected to 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging activity investigation and quantitative structure-activity relationship (QSAR) studies were performed to elucidate their structure-activity relationship. The best multiple linear QSAR model demonstrate a stability in the internal validation and Y-randomization (R² = 0.81; F = 24.225; Q²loo = 0.72; R²Yscr = 0.148). Sphericity of the molecule, ratio of symmetric atoms enhanced atomic mass along the principle axes in regard to total number of atoms in molecule, and 3D distribution of the atoms higher electronegativity (O, N, and S) in molecules are important characteristic for antioxidant ability of rhodanine derivatives. Molecular docking studies were carried out in order to explain in silico antioxidant studies, a specific protein tyrosine kinase (2HCK). The binding interactions of the most active compound have shown strong hydrogen bonding and van der Waals interactions with the target protein.
Subject(s)
Antioxidants/chemistry , Biphenyl Compounds/antagonists & inhibitors , Choline/chemistry , Picrates/antagonists & inhibitors , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Rhodanine/chemistry , Urea/chemistry , Aldehydes/chemistry , Biphenyl Compounds/chemistry , Humans , Molecular Docking Simulation , Picrates/chemistry , Proto-Oncogene Proteins c-hck/chemistry , Quantitative Structure-Activity Relationship , Solutions , Solvents , TemperatureABSTRACT
High level of hematopoietic cell kinase (Hck) is associated with drug resistance in chronic myeloid leukemia. Additionally, Hck activity has also been connected with the pathogenesis of HIV-1 and chronic obstructive pulmonary disease. In this study, three-dimensional (3D) QSAR pharmacophore models were generated for Hck based on experimentally known inhibitors. A best pharmacophore model, Hypo1, was developed with high correlation coefficient (0.975), Low RMS deviation (0.60) and large cost difference (49.31), containing three ring aromatic and one hydrophobic aliphatic feature. It was further validated by the test set (r = 0.96) and Fisher's randomization method (95%). Hypo 1 was used as a 3D query for screening the chemical databases, and the hits were further screened by applying Lipinski's rule of five and ADMET properties. Selected hit compounds were subjected to molecular docking to identify binding conformations in the active site. Finally, the appropriate binding modes of final hit compounds were revealed by molecular dynamics (MD) simulations and free energy calculation studies. Hence, we propose the final three hit compounds as virtual candidates for Hck inhibitors.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-hck/chemistry , Databases, Chemical , Drug Resistance, Neoplasm/genetics , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Molecular Dynamics Simulation , Protein Binding/genetics , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Proto-Oncogene Proteins c-hck/genetics , Quantitative Structure-Activity Relationship , User-Computer InterfaceABSTRACT
The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent Hck activity in biochemical assays and potently blocks HIV replication in vitro. In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated Hck that result from Nef binding, as well as the impact of DFP-4AB on these changes. Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by Hck, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain. No changes in hydrogen exchange were observed in the Hck SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding. When HX MS was performed in the presence of DFP-4AB, the effect of Nef on Hck N-lobe dynamics was completely reversed. These results show that constitutive activation of Hck by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure. DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells.
Subject(s)
HIV Infections/drug therapy , HIV Infections/metabolism , HIV-1/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Proto-Oncogene Proteins c-hck/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , Enzyme Activation/drug effects , HIV-1/metabolism , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Conformation/drug effects , Protein Interaction Maps/drug effects , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-hck/chemistry , Virus Replication/drug effects , nef Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors , src Homology DomainsABSTRACT
Flavonoids reduce cardiovascular disease risk through anti-inflammatory, anti-coagulant and anti-platelet actions. One key flavonoid inhibitory mechanism is blocking kinase activity that drives these processes. Flavonoids attenuate activities of kinases including phosphoinositide-3-kinase, Fyn, Lyn, Src, Syk, PKC, PIM1/2, ERK, JNK and PKA. X-ray crystallographic analyses of kinase-flavonoid complexes show that flavonoid ring systems and their hydroxyl substitutions are important structural features for their binding to kinases. A clearer understanding of structural interactions of flavonoids with kinases is necessary to allow construction of more potent and selective counterparts. We examined flavonoid (quercetin, apigenin and catechin) interactions with Src family kinases (Lyn, Fyn and Hck) applying the Sybyl docking algorithm and GRID. A homology model (Lyn) was used in our analyses to demonstrate that high-quality predicted kinase structures are suitable for flavonoid computational studies. Our docking results revealed potential hydrogen bond contacts between flavonoid hydroxyls and kinase catalytic site residues. Identification of plausible contacts indicated that quercetin formed the most energetically stable interactions, apigenin lacked hydroxyl groups necessary for important contacts and the non-planar structure of catechin could not support predicted hydrogen bonding patterns. GRID analysis using a hydroxyl functional group supported docking results. Based on these findings, we predicted that quercetin would inhibit activities of Src family kinases with greater potency than apigenin and catechin. We validated this prediction using in vitro kinase assays. We conclude that our study can be used as a basis to construct virtual flavonoid interaction libraries to guide drug discovery using these compounds as molecular templates.
Subject(s)
Flavonoids/pharmacology , Protein Kinase Inhibitors/pharmacology , src-Family Kinases/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Apigenin/chemistry , Apigenin/pharmacology , Binding Sites , Catechin/chemistry , Catechin/pharmacology , Drug Evaluation, Preclinical/methods , Flavonoids/chemistry , Humans , Molecular Docking Simulation , Protein Domains , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/metabolism , Quercetin/chemistry , Quercetin/pharmacology , Structure-Activity Relationship , src-Family Kinases/chemistry , src-Family Kinases/metabolismABSTRACT
Hematopoietic cell kinase (Hck) is a member of the Src-family of non-receptor tyrosine kinases, which plays many roles in signalling pathways involved in the regulation of cell processes. Hck is expressed in cells of hematopoietic origin, specifically myelomonocytic cells and B lymphocytes. It participates in phagocytosis, adhesion, migration, regulation of protrusion formation on cell membrane, lysosome exocytosis, podosome formation and actin polymerization. More importantly from a medicinal chemistry point of view, high levels of Hck are involved in chronic myeloid leukemia and other hematologic tumors. Furthermore, Hck activity has been associated with virus infections including HIV-1. In particular, Hck is activated by the HIV-1 accessory protein Nef, a multifunctional HIV-1 protein that accelerates progression to AIDS and enhances the infectivity of progeny viruses. Nef binding to Hck leads to kinase activation which is important in AIDS pathogenesis. For these reasons, Hck represents a potentially good therapeutic target for the treatment of both specific cancers and HIV infection. This article summarizes Hck biological activities connected with malignancies and HIV infection, many of which have been only recently reported, and presents an overview of the compounds endowed with Hck inhibitory activity, especially focusing on the medicinal chemistry aspect.
Subject(s)
HIV Infections/drug therapy , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Animals , Drug Discovery , HIV Infections/enzymology , Humans , Neoplasms/enzymology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/metabolismABSTRACT
HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners.
Subject(s)
HIV-1/genetics , Proto-Oncogene Proteins c-hck/chemistry , nef Gene Products, Human Immunodeficiency Virus/chemistry , Binding Sites , Crystallography, X-Ray , Enzyme Activation , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Expression Regulation , HEK293 Cells , HIV-1/metabolism , Host-Pathogen Interactions , Humans , Models, Molecular , Plasmids/chemistry , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Proto-Oncogene Proteins c-hck/genetics , Proto-Oncogene Proteins c-hck/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Signal Transduction , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
The small kinase inhibitor SKF86002 lacks intrinsic fluorescence but becomes fluorescent upon binding to the ATP-binding sites of p38 mitogen-activated protein kinase (p38α). It was found that co-crystals of this compound with various kinases were distinguishable by their strong fluorescence. The co-crystals of SKF86002 with p38α, Pim1, ASK1, HCK and AMPK were fluorescent. Addition of SKF86002, which binds to the ATP site, to the co-crystallization solution of HCK promoted protein stability and thus facilitated the production of crystals that otherwise would not grow in the apo form. It was further demonstrated that the fluorescence of SKF86002 co-crystals can be applied to screen for candidate kinase inhibitors. When a compound binds competitively to the ATP-binding site of a kinase crystallized with SKF86002, it displaces the fluorescent SKF86002 and the crystal loses its fluorescence. Lower fluorescent signals were reported after soaking SKF86002-Pim1 and SKF86002-HCK co-crystals with the inhibitors quercetin, a quinazoline derivative and A-419259. Determination of the SKF86002-Pim1 and SKF86002-HCK co-crystal structures confirmed that SKF86002 interacts with the ATP-binding sites of Pim1 and HCK. The structures of Pim1-SKF86002 crystals soaked with the inhibitors quercetin and a quinazoline derivative and of HCK-SKF86002 crystals soaked with A-419259 were determined. These structures were virtually identical to the deposited crystal structures of the same complexes. A KINOMEscan assay revealed that SKF86002 binds a wide variety of kinases. Thus, for a broad range of kinases, SKF86002 is useful as a crystal marker, a crystal stabilizer and a marker to identify ligand co-crystals for structural analysis.
Subject(s)
Adenosine Triphosphate/chemistry , Fluorescent Dyes/chemistry , Imidazoles/chemistry , Molecular Probes/chemistry , Protein Kinase Inhibitors/chemistry , Thiazoles/chemistry , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/chemistry , AMP-Activated Protein Kinases/genetics , Binding, Competitive , Crystallization , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , High-Throughput Screening Assays , Humans , Ligands , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Kinase Kinase 5/chemistry , MAP Kinase Kinase Kinase 5/genetics , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Mitogen-Activated Protein Kinase 14/chemistry , Mitogen-Activated Protein Kinase 14/genetics , Protein Binding , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/genetics , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Proto-Oncogene Proteins c-pim-1/chemistry , Proto-Oncogene Proteins c-pim-1/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg(null) mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.
Subject(s)
Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/drug effects , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Adult , Aged , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Bone Marrow Transplantation , Crystallography, X-Ray , Drug Resistance, Neoplasm/drug effects , Female , Hematopoiesis/drug effects , Humans , Leukemia, Myeloid, Acute/drug therapy , Male , Mice , Middle Aged , Neoplastic Stem Cells/enzymology , Neoplastic Stem Cells/pathology , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/metabolism , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , RNA, Small Interfering/metabolism , Small Molecule Libraries/pharmacology , Tumor Cells, Cultured , Young AdultABSTRACT
Timeless was originally identified in Drosophila as an essential component of circadian cycle regulation, where its function is tightly controlled at the protein level by tyrosine phosphorylation and subsequent degradation. In mammals, Timeless has also been implicated in circadian rhythms as well as cell cycle control and embryonic development. Here we report that mammalian Timeless is an SH3 domain-binding protein and substrate for several members of the Src protein-tyrosine kinase family, including Fyn, Hck, c-Src and c-Yes. Co-expression of Tim with Fyn or Hck was followed by ubiquitylation and subsequent degradation in human 293T cells. While c-Src and c-Yes also promoted Tim ubiquitylation, in this case ubiquitylation correlated with Tim protein accumulation rather than degradation. Both c-Src and c-Yes selectively promoted modification of Tim through Lys63-linked polyubiquitin, which may explain the differential effects on Tim protein turnover. These data show distinct and opposing roles for individual Src-family members in the regulation of Tim protein levels, suggesting a unique mechanism for the regulation of Tim function in mammals.
Subject(s)
Cell Cycle Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , src-Family Kinases/metabolism , Animals , CSK Tyrosine-Protein Kinase , Cell Cycle Proteins/genetics , Drosophila Proteins , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Proto-Oncogene Proteins c-fyn/chemistry , Proto-Oncogene Proteins c-fyn/genetics , Proto-Oncogene Proteins c-fyn/metabolism , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/genetics , Proto-Oncogene Proteins c-hck/metabolism , Proto-Oncogene Proteins c-yes/chemistry , Proto-Oncogene Proteins c-yes/genetics , Proto-Oncogene Proteins c-yes/metabolism , Substrate Specificity , Transfection , Ubiquitination , src Homology Domains , src-Family Kinases/chemistry , src-Family Kinases/geneticsABSTRACT
The functions of Src family kinases are tightly regulated through Src homology (SH) domain-mediated protein-protein interactions. We previously reported the biophysical characteristics of the apoptosis-linked gene 2-interacting protein X (Alix) in complex with the haemopoietic cell kinase (Hck) SH3 domain. In the current study, we have combined ITC, NMR, SAXS and molecular modeling to determine a 3D model of the complex. We demonstrate that Hck SH3 recognizes an extended linear proline-rich region of Alix. This particular binding mode enables Hck SH3 to sense a specific non-canonical residue situated in the SH3 RT-loop of the kinase. The resulting model helps clarify the mechanistic insights of Alix-Hck interaction.
Subject(s)
Calcium-Binding Proteins/chemistry , Cell Cycle Proteins/chemistry , Endosomal Sorting Complexes Required for Transport/chemistry , Proto-Oncogene Proteins c-hck/chemistry , src Homology Domains , Binding Sites , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Proline/genetics , Proline/metabolism , Protein Conformation , Proto-Oncogene Proteins c-hck/metabolism , Scattering, Small Angle , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Signaling cascades are managed in time and space by interactions between and among proteins. These interactions are often aided by adaptor proteins, which guide enzyme-substrate pairs into proximity. Miniature proteins are a class of small, well-folded protein domains possessing engineered binding properties. Here we made use of two miniature proteins with complementary binding properties to create a synthetic adaptor protein that effectively redirects a ubiquitous signaling event: tyrosine phosphorylation. We report that miniature-protein-based adaptor 3 uses templated catalysis to redirect the Src family kinase Hck to phosphorylate hDM2, a negative regulator of the p53 tumor suppressor and a poor Hck substrate. Phosphorylation occurs with multiple turnover and at a single site targeted by c-Abl kinase in the cell.
Subject(s)
Proto-Oncogene Proteins c-hck/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Humans , Models, Molecular , Phosphorylation , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-mdm2/chemistry , Substrate Specificity , Tyrosine/metabolismABSTRACT
Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef(+) wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , HIV-1/pathogenicity , nef Gene Products, Human Immunodeficiency Virus/metabolism , Amino Acid Motifs , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Drug Evaluation, Preclinical , HEK293 Cells , HIV-1/metabolism , Humans , Models, Molecular , Proline , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/metabolism , nef Gene Products, Human Immunodeficiency Virus/chemistry , src Homology DomainsABSTRACT
Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.
Subject(s)
Computational Biology/methods , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/metabolism , Amino Acid Motifs , Amino Acid Sequence , CD4 Antigens/chemistry , CD4 Antigens/metabolism , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , HLA-A Antigens/chemistry , HLA-A Antigens/metabolism , Humans , Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 1/metabolism , Molecular Sequence Data , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Protein Binding , Proto-Oncogene Proteins c-fyn/chemistry , Proto-Oncogene Proteins c-fyn/metabolism , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/metabolism , Proto-Oncogene Proteins c-vav/chemistry , Proto-Oncogene Proteins c-vav/metabolism , Receptors for Activated C Kinase , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolismABSTRACT
The human immunodeficiency virus 1 (HIV-1) Nef protein is a pathogenicity factor required for effective progression to AIDS, which modulates host cell signaling pathways and T-cell receptor internalization. We have determined the crystal structure of Nef, allele SF2, in complex with an engineered SH3 domain of human Hck showing unnaturally tight binding and inhibitory potential toward Nef. This complex provides the most complete Nef structure described today, and explains the structural basis of the high affinity of this interaction. Intriguingly, the 33-residue C-terminal flexible loop is resolved in the structure by its interactions with a highly conserved hydrophobic groove on the core domain of an adjacent Nef molecule. The loop mediates the interaction of Nef with the cellular adaptor protein machinery for the stimulated internalization of surface receptors. The endocytic dileucine-based sorting motif is exposed at the tip of the acidic loop, giving the myristoylated Nef protein a distinctly dipolar character. The intermolecular domain assembly of Nef provides insights into a possible regulation mechanism for cargo trafficking.