ABSTRACT
The evolutionarily related deubiquitinating enzymes (DUBs) USP25 and USP28 comprise an identical overall domain architecture but are functionally non-redundant: USP28 stabilizes c-MYC and other nuclear proteins, and USP25 regulates inflammatory TRAF signaling. We here compare molecular features of USP25 and USP28. Active enzymes form distinctively shaped dimers, with a dimerizing insertion spatially separating independently active catalytic domains. In USP25, but not USP28, two dimers can form an autoinhibited tetramer, where a USP25-specific, conserved insertion sequence blocks ubiquitin binding. In full-length enzymes, a C-terminal domain with a previously unknown fold has no impact on oligomerization, but N-terminal regions affect the dimer-tetramer equilibrium in vitro. We confirm oligomeric states of USP25 and USP28 in cells and show that modulating oligomerization affects substrate stabilization in accordance with in vitro activity data. Our work highlights how regions outside of the catalytic domain enable a conceptually intriguing interplay of DUB oligomerization and activity.
Subject(s)
Inflammation/genetics , Protein Conformation , Ubiquitin Thiolesterase/genetics , Amino Acid Sequence/genetics , Catalytic Domain/genetics , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/genetics , Humans , Inflammation/pathology , Mutation/genetics , Protein Binding/genetics , Protein Domains/genetics , Protein Multimerization/genetics , Proto-Oncogene Proteins c-myb/chemistry , Proto-Oncogene Proteins c-myb/genetics , Signal Transduction/genetics , Substrate Specificity , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/genetics , Ubiquitin/genetics , Ubiquitin Thiolesterase/chemistryABSTRACT
Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al], [Nature] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al], [Nature] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [J. Biol. Chem. ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al], [Nature] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al], [Nat. Struct. Mol. Biol.] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.
Subject(s)
Carbon-Nitrogen Ligases/metabolism , Protein Isoforms/metabolism , Cell Proliferation/physiology , Humans , Immunologic Deficiency Syndromes/metabolism , T-Lymphocytes/metabolismABSTRACT
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.
Subject(s)
Piperidines/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Animals , Apoenzymes/antagonists & inhibitors , Apoenzymes/chemistry , Apoenzymes/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Female , Humans , Mice , Models, Molecular , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/pathology , Piperidines/chemical synthesis , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/metabolism , Pyrazoles/chemical synthesis , Pyrimidines/chemical synthesis , Substrate Specificity , Transcription, Genetic/drug effects , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Specific Peptidase 7/chemistry , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitination/drug effects , Xenograft Model Antitumor AssaysABSTRACT
TYK2 is a member of the JAK family of kinases and a key mediator of IL-12, IL-23, and type I interferon signaling. These cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genetic association studies, TYK2 inhibition is an attractive therapeutic strategy for these diseases. Herein, we report the discovery of a series of highly selective catalytic site TYK2 inhibitors designed using FEP+ and structurally enabled design starting from a virtual screen hit. We highlight the structure-based optimization to identify a lead candidate 30, a potent cellular TYK2 inhibitor with excellent selectivity, pharmacokinetic properties, and in vivo efficacy in a mouse psoriasis model.
Subject(s)
Psoriasis , TYK2 Kinase , Animals , Humans , Janus Kinases , Mice , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Psoriasis/drug therapy , RodentiaABSTRACT
The discovery, structure-activity relationships, and optimization of a novel class of fatty acid synthase (FASN) inhibitors is reported. High throughput screening identified a series of substituted piperazines with structural features that enable interactions with many of the potency-driving regions of the FASN KR domain binding site. Derived from this series was FT113, a compound with potent biochemical and cellular activity, which translated into excellent activity in in vivo models.
Subject(s)
Fatty Acid Synthases/antagonists & inhibitors , Piperazines/chemistry , Administration, Oral , Animals , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Evaluation, Preclinical , Fatty Acid Synthases/metabolism , Half-Life , Humans , Malonyl Coenzyme A/metabolism , Mice , Mice, Nude , Molecular Docking Simulation , Neoplasms/drug therapy , Neoplasms/metabolism , Piperazines/administration & dosage , Piperazines/pharmacokinetics , Piperazines/pharmacology , Protein Structure, Tertiary , Rats , Structure-Activity RelationshipABSTRACT
Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.
Subject(s)
Actins/metabolism , Drosophila Proteins/metabolism , Microfilament Proteins/metabolism , Models, Molecular , Nerve Tissue Proteins/chemistry , Oogenesis/physiology , Protein Conformation , Protein Structure, Tertiary/genetics , Animals , Crystallization , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster , Fluorescence Polarization , Humans , Microfilament Proteins/chemistry , Microfilament Proteins/geneticsABSTRACT
TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30, a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.
Subject(s)
Arthritis, Rheumatoid , Autoimmune Diseases , Psoriasis , Humans , TYK2 Kinase , Genome-Wide Association Study , Autoimmune Diseases/drug therapy , Psoriasis/drug therapyABSTRACT
Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the "gatekeeper" residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a "generic" resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.
Subject(s)
Adenosine Triphosphate/metabolism , Drug Resistance/genetics , ErbB Receptors/genetics , Mutation , Animals , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Gefitinib , Insecta , Kinetics , Protein Conformation , Quinazolines/pharmacologyABSTRACT
Inhibition of mutant IDH1 is being evaluated clinically as a treatment option for oncology. Here we describe the structure-based design and optimization of quinoline lead compounds to identify FT-2102, a potent, orally bioavailable, brain penetrant, and selective mIDH1 inhibitor. FT-2102 has excellent ADME/PK properties and reduces 2-hydroxyglutarate levels in an mIDH1 xenograft tumor model. This compound has been selected as a candidate for clinical development in hematologic malignancies, solid tumors, and gliomas with mIDH1.
Subject(s)
Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Isocitrate Dehydrogenase/antagonists & inhibitors , Neoplasms/drug therapy , Pyridines/therapeutic use , Quinolines/therapeutic use , Quinolones/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Cell Line, Tumor , Drug Design , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Female , Humans , Isocitrate Dehydrogenase/metabolism , Mice, Inbred BALB C , Molecular Structure , Protein Binding , Pyridines/chemical synthesis , Pyridines/metabolism , Quinolines/chemical synthesis , Quinolines/metabolism , Quinolones/chemical synthesis , Quinolones/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Mutations at the arginine residue (R132) in isocitrate dehydrogenase 1 (IDH1) are frequently identified in various human cancers. Inhibition of mutant IDH1 (mIDH1) with small molecules has been clinically validated as a promising therapeutic treatment for acute myeloid leukemia and multiple solid tumors. Herein, we report the discovery and optimization of a series of quinolinones to provide potent and orally bioavailable mIDH1 inhibitors with selectivity over wild-type IDH1. The X-ray structure of an early lead 24 in complex with mIDH1-R132H shows that the inhibitor unexpectedly binds to an allosteric site. Efforts to improve the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties of 24 yielded a preclinical candidate 63. The detailed preclinical ADME and pharmacology studies of 63 support further development of quinolinone-based mIDH1 inhibitors as therapeutic agents in human trials.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Isocitrate Dehydrogenase/antagonists & inhibitors , Quinolones/chemistry , Quinolones/pharmacology , Allosteric Site/drug effects , Animals , Biological Availability , Cell Line, Tumor , Crystallography, X-Ray , Dogs , Drug Discovery , Enzyme Inhibitors/pharmacokinetics , Female , Humans , Isocitrate Dehydrogenase/chemistry , Isocitrate Dehydrogenase/genetics , Madin Darby Canine Kidney Cells , Mice , Mice, Inbred BALB C , Models, Molecular , Point Mutation , Quinolones/pharmacokineticsABSTRACT
Jak-family tyrosine kinases mediate signaling from diverse cytokine receptors. Binding of Jaks to their cognate receptors is mediated by their N-terminal region, which contains FERM and SH2 domains. Here we describe the crystal structure of the FERM-SH2 region of Jak2 at 3.0Å resolution. The structure reveals that these domains and their flanking linker segments interact intimately to form an integrated structural module. The Jak2 FERM-SH2 structure closely resembles that recently described for Tyk2, another member of the Jak family. While the overall architecture and interdomain orientations are preserved between Jak2 and Tyk2, we identify residues in the putative receptor-binding groove that differ between the two and may contribute to the specificity of receptor recognition. Analysis of Jak mutations that are reported to disrupt receptor binding reveals that they lie in the hydrophobic core of the FERM domain, and are thus expected to compromise the structural integrity of the FERM-SH2 unit. Similarly, analysis of mutations in Jak3 that are associated with severe combined immunodeficiency suggests that they compromise Jak3 function by destabilizing the FERM-SH2 structure.
Subject(s)
Janus Kinase 2/chemistry , Protein Conformation , src Homology Domains , Crystallography, X-Ray , Humans , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Models, Molecular , Mutation/genetics , Phosphorylation , Protein Binding , Signal TransductionABSTRACT
The V617F mutation in the Jak2 pseudokinase domain causes myeloproliferative neoplasms, and the equivalent mutation in Jak1 (V658F) is found in T-cell leukemias. Crystal structures of wild-type and V658F-mutant human Jak1 pseudokinase reveal a conformational switch that remodels a linker segment encoded by exon 12, which is also a site of mutations in Jak2. This switch is required for V617F-mediated Jak2 activation and possibly for physiologic Jak activation.
Subject(s)
Janus Kinases/metabolism , Oncogenes , Enzyme Activation , Humans , Janus Kinases/chemistry , Models, Molecular , Protein ConformationABSTRACT
Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1) induces type I interferon expression and modulates nuclear factor κB (NF-κB) signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ε (IKKε) but not in IKKß, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.
Subject(s)
Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Serine-Threonine Kinases/chemistry , Ubiquitination , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Humans , I-kappa B Kinase/metabolism , Interferon Regulatory Factor-3/metabolism , Molecular Sequence Data , Protein Binding , Protein Multimerization , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein Structure, TertiaryABSTRACT
Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor-like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100-1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors.
Subject(s)
HSP90 Heat-Shock Proteins/antagonists & inhibitors , Isoxazoles/pharmacology , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/genetics , Leukemia, B-Cell/enzymology , Myeloproliferative Disorders/enzymology , Resorcinols/pharmacology , Signal Transduction/physiology , Animals , Cell Line, Tumor , Cell Proliferation , DNA Primers/genetics , Female , Flow Cytometry , Gene Expression Profiling , HSP90 Heat-Shock Proteins/metabolism , Humans , Immunoblotting , Immunohistochemistry , Isoxazoles/therapeutic use , Janus Kinase 2/metabolism , Leukemia, B-Cell/drug therapy , Leukemia, B-Cell/genetics , Luciferases , Mice , Mice, Inbred BALB C , Mutagenesis , Mutation, Missense/genetics , Myeloproliferative Disorders/drug therapy , Myeloproliferative Disorders/genetics , Phosphorylation , RNA, Small Interfering/genetics , Receptors, Cytokine/genetics , Receptors, Cytokine/metabolism , Resorcinols/therapeutic use , X-Ray MicrotomographyABSTRACT
The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535-700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.
Subject(s)
rho-Associated Kinases/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Dimerization , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino Acid , Tropomyosin/chemistryABSTRACT
Aurora kinases are key regulators of cell division and important targets for cancer therapy. We report that Binucleine 2 is a highly isoform-specific inhibitor of Drosophila Aurora B kinase, and we identify a single residue within the kinase active site that confers specificity for Aurora B. Using Binucleine 2, we show that Aurora B kinase activity is not required during contractile ring ingression, providing insight into the mechanism of cytokinesis.
Subject(s)
Cell Cycle Proteins/physiology , Cytokinesis/physiology , Drosophila Proteins/physiology , Drosophila melanogaster/cytology , Drosophila melanogaster/enzymology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/physiology , Pyrazoles/chemistry , Schiff Bases/chemistry , Amino Acid Sequence , Animals , Aurora Kinase B , Aurora Kinases , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Cytokinesis/drug effects , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/chemistry , Drosophila melanogaster/drug effects , Isoenzymes/antagonists & inhibitors , Isoleucine/chemistry , Molecular Sequence Data , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistry , Pyrazoles/pharmacology , RNA Interference , Schiff Bases/pharmacologyABSTRACT
Bacillus subtilis gene products TenA and TenI have been implicated in regulating the production of extracellular proteases, but their role in the regulation process remains unclear. The structural characterization of these proteins was undertaken to help provide insight into their function. We have determined the structure of TenA alone and in complex with 4-amino-2-methyl-5-hydroxymethylpyrimidine, and we demonstrate that TenA is a thiaminase II. The TenA structure suggests that the degradation of thiamin by TenA likely proceeds via the same addition-elimination mechanism described for thiaminase I. Three active-site residues, Asp44, Cys135, and Glu205, are likely involved in substrate binding and catalysis based on the enzyme/product complex structure and the conservation of these residues within TenA sequences. We have also determined the structure of TenI. Although TenI shows significant structural homology to thiamin phosphate synthase, it has no known enzymatic function. The structure suggests that TenI is unable to bind thiamin phosphate, largely resulting from the presence of leucine at position 119, while the corresponding residue in thiamin phosphate synthase is glycine.
Subject(s)
Bacillus subtilis/chemistry , Bacillus subtilis/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Hydrolases/chemistry , Repressor Proteins/chemistry , Trans-Activators/chemistry , Alkyl and Aryl Transferases/chemistry , Binding Sites , Crystallization , Crystallography, X-Ray , Dimerization , Hydrolases/metabolism , Protein Structure, Quaternary , Protein Structure, Secondary , Pyrimidines/metabolism , Repressor Proteins/metabolism , Sulfates/metabolism , Thiamine Triphosphate/chemistry , Thiamine Triphosphate/metabolism , Trans-Activators/metabolismABSTRACT
In an effort to develop potent multisubstrate-analog inhibitors of purine nucleoside phosphorylase (PNP), three nucleoside phosphonates were designed utilizing structural information from the previously reported structures of complexes of bovine PNP with substrates and products. The nucleoside phosphonates contain an acetal linkage at the O2' and O3' positions and a two-C-atom spacer between the ribose and phosphate moieties. The linkage enables the compounds to simultaneously occupy the purine-, ribose- and phosphate-binding sites. The chemical syntheses, inhibition profiles and structural characterization of these novel multisubstrate analog inhibitors with bovine PNP are described.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Nucleosides/chemistry , Nucleosides/pharmacology , Organophosphonates/chemistry , Organophosphonates/pharmacology , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Animals , Binding Sites , Cattle , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Inhibitory Concentration 50 , Inosine/metabolism , Models, Molecular , Nucleosides/chemical synthesis , Organophosphonates/chemical synthesis , Purine-Nucleoside Phosphorylase/chemistry , Purine-Nucleoside Phosphorylase/metabolism , Structure-Activity RelationshipABSTRACT
S-adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine biosynthetic pathway and belongs to a small class of pyruvoyl-dependent amino acid decarboxylases. Structural elucidation of the prokaryotic AdoMetDC is of substantial interest in order to determine the relationship between the eukaryotic and prokaryotic forms of the enzyme. Although both forms utilize pyruvoyl groups, there is no detectable sequence similarity except at the site of pyruvoyl group formation. The x-ray structure of the Thermatoga maritima AdoMetDC proenzyme reveals a dimeric protein fold that is remarkably similar to the eukaryotic AdoMetDC protomer, suggesting an evolutionary link between the two forms of the enzyme. Three key active site residues (Ser55, His68, and Cys83) involved in substrate binding, catalysis or proenzyme processing that were identified in the human and potato AdoMet-DCs are structurally conserved in the T. maritima AdoMetDC despite very limited primary sequence identity. The role of Ser55, His68, and Cys83 in the self-processing reaction was investigated through site-directed mutagenesis. A homology model for the Escherichia coli AdoMetDC was generated based on the structures of the T. maritima and human AdoMetDCs.