ABSTRACT
Invasive fungal infections have mortality rates of 30-90%, depending on patient co-morbidities and the causative pathogen. The frequent emergence of drug resistance reduces the efficacy of currently approved treatment options, highlighting an urgent need for antifungals with new modes of action. Addressing this need, fosmanogepix (N-phosphonooxymethylene prodrug of manogepix; MGX) is the first in a new class of gepix drugs, and acts as a broad-spectrum, orally bioavailable inhibitor of the essential fungal glycosylphosphatidylinositol (GPI) acyltransferase Gwt1. MGX inhibits the growth of diverse fungal pathogens and causes accumulation of immature GPI-anchored proteins in the fungal endoplasmic reticulum. Relevant to the ongoing clinical development of fosmanogepix, we report a synergistic, fungicidal interaction between MGX and inhibitors of the protein phosphatase calcineurin against important human fungal pathogens. To investigate this synergy further, we evaluated a library of 124 conditional expression mutants covering 95% of the genes encoding proteins involved in GPI-anchor biosynthesis or proteins predicted to be GPI-anchored. Strong negative chemical-genetic interactions between the calcineurin inhibitor FK506 and eleven GPI-anchor biosynthesis genes were identified, indicating that calcineurin signalling is required for fungal tolerance to not only MGX, but to inhibition of the GPI-anchor biosynthesis pathway more broadly. Depletion of these GPI-anchor biosynthesis genes, like MGX treatment, also exposed fungal cell wall (1â3)-ß-D-glucans. Taken together, these findings suggest the increased risk of invasive fungal infections associated with use of calcineurin inhibitors as immunosuppressants may be mitigated by their synergistic fungicidal interaction with (fos)manogepix and its ability to enhance exposure of immunostimulatory glucans.
ABSTRACT
Invasive aspergillosis (IA) due to Aspergillus fumigatus is a deadly infection for which new antifungal therapies are needed. Here, we demonstrate the efficacy of a Gwt1 inhibitor, APX2041, and its prodrug, APX2104, against A. fumigatus. The wild-type, azole-resistant, and echinocandin-resistant A. fumigatus strains were equally susceptible to APX2041 in vitro. APX2104 treatment in vivo significantly prolonged survival of neutropenic mice challenged with the wild-type and azole-resistant strains, revealing APX2104 as a potentially promising therapeutic against IA.
Subject(s)
Aspergillus fumigatus , Prodrugs , Animals , Antifungal Agents/pharmacology , Antifungal Agents/therapeutic use , Drug Resistance, Fungal/genetics , Isoxazoles , Mice , Microbial Sensitivity Tests , Prodrugs/pharmacologyABSTRACT
An ongoing Candida auris outbreak in the New York metropolitan area is the largest recorded to date in North America. Laboratory surveillance revealed NY C. auris isolates are resistant to fluconazole, with variable resistance to other currently used broad-spectrum antifungal drugs, and that several isolates are panresistant. Thus, there is an urgent need for new drugs with a novel mechanism of action to combat the resistance challenge. Manogepix (MGX) is a first-in-class agent that targets the fungal Gwt1 enzyme. The prodrug fosmanogepix is currently in phase 2 clinical development for the treatment of fungal infections. We evaluated the susceptibility of 200 New York C. auris isolates to MGX and 10 comparator drugs using CLSI methodology. MGX demonstrated lower MICs than comparators (MIC50 and MIC90, 0.03 mg/liter; range, 0.004 to 0.06 mg/liter). The local epidemiological cutoff value (ECV) for MGX indicated all C. auris isolates were within the population of wild-type (WT) strains; 0.06 mg/liter defines the upper limit of wild type (UL-WT). MGX was 8- to 32-fold more active than the echinocandins, 16- to 64-fold more active than the azoles, and 64-fold more active than amphotericin B. No differences were found in the MGX or comparators' MIC50, MIC90, or geometric mean (GM) values when subsets of clinical, surveillance, and environmental isolates were evaluated. The range of MGX MIC values for six C. auris panresistant isolates was 0.008 to 0.015 mg/liter, and the median and mode MIC values were 0.015 mg/liter, demonstrating that MGX retains activity against these isolates. These data support further clinical evaluation of fosmanogepix for the treatment of C. auris infections, including highly resistant isolates.
Subject(s)
Antifungal Agents , Candida , Aminopyridines , Antifungal Agents/pharmacology , Disease Outbreaks , Isoxazoles , Microbial Sensitivity Tests , New York , North AmericaABSTRACT
Manogepix is a broad-spectrum antifungal agent that inhibits glycosylphosphatidylinositol (GPI) anchor biosynthesis. Using whole-genome sequencing, we characterized two efflux-mediated mechanisms in the fungal pathogens Candida albicans and Candida parapsilosis that resulted in decreased manogepix susceptibility. In C. albicans, a gain-of-function mutation in the transcription factor gene ZCF29 activated expression of ATP-binding cassette transporter genes CDR11 and SNQ2 In C. parapsilosis, a mitochondrial deletion activated expression of the major facilitator superfamily transporter gene MDR1.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Aminopyridines/pharmacology , Antifungal Agents/pharmacology , Candida albicans/genetics , Candida parapsilosis/genetics , Isoxazoles/pharmacology , Biological Transport/genetics , Candida albicans/drug effects , Candida albicans/metabolism , Candida parapsilosis/drug effects , Candida parapsilosis/metabolism , Drug Resistance, Fungal/genetics , Genome, Fungal/genetics , Microbial Sensitivity Tests , Whole Genome SequencingABSTRACT
Fosmanogepix (APX001) is a first-in-class prodrug molecule that is currently in Phase 2 clinical trials for invasive fungal infections. The active moiety manogepix (APX001A) inhibits the novel fungal protein Gwt1. Gwt1 catalyzes an early step in the GPI anchor biosynthesis pathway. Here we describe the synthesis and evaluation of 292 new and 24 previously described analogs that were synthesized using a series of advanced intermediates to allow for rapid analoging. Several compounds demonstrated significantly (8- to 32-fold) improved antifungal activity against both Cryptococcus neoformans and C. gattii as compared to manogepix. Further in vitro characterization identified three analogs with a similar preliminary safety and in vitro profile to manogepix and superior activity against Cryptococcus spp.
Subject(s)
Aminopyridines/pharmacology , Antifungal Agents/pharmacology , Cryptococcus/drug effects , Isoxazoles/pharmacology , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Aminopyridines/chemical synthesis , Aminopyridines/chemistry , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Dose-Response Relationship, Drug , Fungal Proteins , Isoxazoles/chemical synthesis , Isoxazoles/chemistry , Microbial Sensitivity Tests , Molecular Structure , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Manogepix (MGX) targets the conserved fungal Gwt1 enzyme required for acylation of inositol early in the glycosylphosphatidylinositol biosynthesis pathway. The prodrug fosmanogepix is currently in clinical development for the treatment of invasive fungal infections. We determined that the median frequencies of spontaneous mutations conferring reduced susceptibility to MGX in Candida albicans, C. glabrata, and C. parapsilosis ranged from 3 × 10-8 to <1.85 × 10-8 Serial passage on agar identified mutants of C. albicans and C. parapsilosis with reduced susceptibility to MGX; however, this methodology did not result in C. glabrata mutants with reduced susceptibility. Similarly, serial passage in broth resulted in ≤2-fold changes in population MIC values for C. tropicalis, C. auris, and C. glabrata A spontaneous V163A mutation in the Gwt1 protein of C. glabrata and a corresponding C. albicans heterozygous V162A mutant were obtained. A C. glabrata V163A Gwt1 mutant generated using CRISPR, along with V162A and V168A mutants expressed in C. albicans and Saccharomyces cerevisiae Gwt1, respectively, all demonstrated reduced susceptibility to MGX versus control strains, suggesting the importance of this valine residue to MGX binding across different species. Cross-resistance to the three major classes of antifungals was evaluated, but no changes in susceptibility to amphotericin B or caspofungin were observed in any mutant. No change was observed in fluconazole susceptibility, with the exception of a single non-Gwt1 mutant, where a 4-fold increase in the fluconazole MIC was observed. MGX demonstrated a relatively low potential for resistance development, consistent with other approved antifungal agents and those in clinical development.
Subject(s)
Aminopyridines/pharmacology , Antifungal Agents/pharmacology , Candida/drug effects , Isoxazoles/pharmacology , Acyltransferases/chemistry , Acyltransferases/genetics , Amino Acid Sequence , Biosynthetic Pathways/drug effects , Biosynthetic Pathways/genetics , Candida/genetics , Candida/metabolism , Candidiasis/drug therapy , Candidiasis/microbiology , Drug Resistance, Fungal/genetics , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Fungal , Glycosylphosphatidylinositols/biosynthesis , Glycosylphosphatidylinositols/chemistry , Glycosylphosphatidylinositols/genetics , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microbial Sensitivity Tests , Mutation , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
Invasive pulmonary aspergillosis (IPA) due to Aspergillus fumigatus is a serious fungal infection in the immunosuppressed patient population. Despite the introduction of new antifungal agents, mortality rates remain high, and new treatments are needed. The novel antifungal APX001A targets the conserved Gwt1 enzyme required for the localization of glycosylphosphatidylinositol-anchored mannoproteins in fungi. We evaluated the in vitro activity of APX001A against A. fumigatus and the in vivo activity of its prodrug APX001 in an immunosuppressed mouse model of IPA. APX001A inhibited the growth of A. fumigatus with a minimum effective concentration of 0.03 µg/ml. The use of 50 mg/kg 1-aminobenzotriazole (ABT), a suicide inhibitor of cytochrome P450 enzymes, enhanced APX001A exposures (area under the time-concentration curve [AUC]) 16- to 18-fold and enhanced serum half-life from â¼1 to 9 h, more closely mimicking human pharmacokinetics. We evaluated the efficacy of APX001 (with ABT) in treating murine IPA compared to posaconazole treatment. Treatment of mice with 78 mg/kg once daily (QD), 78 mg/kg twice daily, or 104 mg/kg QD APX001 significantly enhanced the median survival time and prolonged day 21 postinfection overall survival compared to the placebo. Furthermore, administration of APX001 resulted in a significant reduction in lung fungal burden (4.2 to 7.6 log10 conidial equivalents/g of tissue) versus the untreated control and resolved the infection, as judged by histopathological examination. The observed survival and tissue clearance were comparable to a clinically relevant posaconazole dose. These results warrant the continued development of APX001 as a broad-spectrum, first-in-class treatment of invasive fungal infections.
Subject(s)
Aminopyridines/pharmacology , Aminopyridines/therapeutic use , Antifungal Agents/pharmacology , Antifungal Agents/therapeutic use , Invasive Pulmonary Aspergillosis/drug therapy , Isoxazoles/pharmacology , Isoxazoles/therapeutic use , Animals , Disease Models, Animal , Fungal Proteins/genetics , Fungal Proteins/metabolism , Immunocompromised Host , Invasive Pulmonary Aspergillosis/microbiology , Male , Mice , Mice, Inbred ICR , Microbial Sensitivity Tests , Triazoles/therapeutic useABSTRACT
Coccidioidomycosis is a systemic fungal infection caused by the inhalation of the arthroconidia of either of two closely related dimorphic fungi, Coccidioides immitis and C. posadasii, that are endemic in the southwestern United States and other areas in the Western Hemisphere. Chronic cavitary pulmonary infections and extrapulmonary sites of infection are very difficult to treat and often require lifelong azole therapy. APX001A is the first in a new class of broad-spectrum antifungal agents that inhibit Gwt1, an enzyme which is required for cell wall localization of glycosylphosphatidylinositol (GPI)-anchored mannoproteins in fungi. APX001A and several analogs were highly active against clinical isolates of Coccidioides, inhibiting hyphal growth at low nanogram/ml concentrations. APX001 is the N-phosphonooxymethyl prodrug of APX001A, currently in clinical trials for the treatment of invasive fungal infections. Mice were treated orally once daily with 26 mg/kg/day of APX001 and the prodrug analog APX2097, 2 h after administration of the pan-cytochrome P450 inhibitor 1-aminobenzotriazole, which was used to enhance drug half-life and exposures to more closely mimic human pharmacokinetics of APX001A. Five days of treatment reduced lung colony counts by nearly 3 logs and prevented dissemination, similar to the efficacy of fluconazole dosed orally at 25 mg/kg twice daily. In a survival experiment, both APX001- and APX2097-treated mice survived significantly longer than control and fluconazole-treated mice. APX001 and other members of this new class of antifungal agents may offer great promise as effective therapies for coccidioidomycosis.
Subject(s)
Aminopyridines/therapeutic use , Antifungal Agents/therapeutic use , Coccidioides/pathogenicity , Isoxazoles/therapeutic use , Pneumonia/drug therapy , Amphotericin B/therapeutic use , Animals , Coccidioides/drug effects , Disease Models, Animal , Female , Fluconazole/therapeutic use , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Prodrugs/therapeutic use , Triazoles/therapeutic useABSTRACT
Cryptococcal meningitis (CM), caused primarily by Cryptococcus neoformans, is uniformly fatal if not treated. Treatment options are limited, especially in resource-poor geographical regions, and mortality rates remain high despite current therapies. Here we evaluated the in vitro and in vivo activity of several compounds, including APX001A and its prodrug, APX001, currently in clinical development for the treatment of invasive fungal infections. These compounds target the conserved Gwt1 enzyme that is required for the localization of glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins in fungi. The Gwt1 inhibitors had low MIC values, ranging from 0.004 µg/ml to 0.5 µg/ml, against both C. neoformans and C. gattii APX001A and APX2020 demonstrated in vitro synergy with fluconazole (fractional inhibitory concentration index, 0.37 for both). In a CM model, APX001 and fluconazole each alone reduced the fungal burden in brain tissue (0.78 and 1.04 log10 CFU/g, respectively), whereas the combination resulted in a reduction of 3.52 log10 CFU/g brain tissue. Efficacy, as measured by a reduction in the brain and lung tissue fungal burden, was also observed for another Gwt1 inhibitor prodrug, APX2096, where dose-dependent reductions in the fungal burden ranged from 5.91 to 1.79 log10 CFU/g lung tissue and from 7.00 and 0.92 log10 CFU/g brain tissue, representing the nearly complete or complete sterilization of lung and brain tissue at the higher doses. These data support the further clinical evaluation of this new class of antifungal agents for the treatment of CM.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Aminopyridines/pharmacology , Antifungal Agents/pharmacology , Cryptococcus neoformans/drug effects , Fungal Proteins/antagonists & inhibitors , Isoxazoles/pharmacology , Meningitis, Cryptococcal/drug therapy , Organophosphates/pharmacology , Prodrugs/pharmacology , Administration, Oral , Amidohydrolases/genetics , Amidohydrolases/metabolism , Aminopyridines/chemical synthesis , Aminopyridines/pharmacokinetics , Animals , Antifungal Agents/chemical synthesis , Antifungal Agents/pharmacokinetics , Brain/drug effects , Brain/microbiology , Cryptococcus gattii/drug effects , Cryptococcus gattii/enzymology , Cryptococcus gattii/genetics , Cryptococcus gattii/growth & development , Cryptococcus neoformans/enzymology , Cryptococcus neoformans/genetics , Cryptococcus neoformans/growth & development , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Combinations , Drug Synergism , Fluconazole/pharmacology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Injections, Intraperitoneal , Isoxazoles/chemical synthesis , Isoxazoles/pharmacokinetics , Lung/drug effects , Lung/microbiology , Male , Meningitis, Cryptococcal/microbiology , Mice , Microbial Sensitivity Tests , Organophosphates/chemical synthesis , Organophosphates/pharmacokinetics , Prodrugs/chemical synthesis , Prodrugs/pharmacokineticsABSTRACT
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies.
Subject(s)
Immunoglobulin Fab Fragments/chemistry , Receptor, Serotonin, 5-HT2B/chemistry , Receptor, Serotonin, 5-HT2B/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Binding, Competitive , Catalytic Domain , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/metabolism , Ergotamine/chemistry , Ergotamine/metabolism , Humans , Immunoglobulin Fab Fragments/metabolism , Models, Molecular , Protein Conformation , Receptor, Serotonin, 5-HT2B/metabolism , Serotonin Receptor Agonists/chemistry , Serotonin Receptor Agonists/metabolismABSTRACT
A novel antifungal strategy targeting the inhibition of calcineurin is described. To develop a calcineurin based inhibitor of pathogenic fungi, analogs of FK506 were synthesized that were able to permeate mammalian but not fungal cells. Antagonists in combination with FK506 were not immunosuppressive and retained antifungal activity in A. fumigatus. To reduce the dosage burden of the antagonist, murine oral PK was improved an order of magnitude relative to previous FK506 antagonists.
Subject(s)
Antifungal Agents/pharmacology , Calcineurin Inhibitors/pharmacology , Tacrolimus/analogs & derivatives , Tacrolimus/pharmacology , Animals , Antifungal Agents/chemical synthesis , Antifungal Agents/pharmacokinetics , Antifungal Agents/toxicity , Aspergillus fumigatus/drug effects , Calcineurin Inhibitors/chemical synthesis , Calcineurin Inhibitors/pharmacokinetics , Calcineurin Inhibitors/toxicity , Chlorocebus aethiops , Hep G2 Cells , Humans , Interleukin-2/metabolism , Jurkat Cells , Tacrolimus/chemical synthesis , Tacrolimus/pharmacokinetics , Tacrolimus/toxicity , Tacrolimus Binding Protein 1A/chemistry , Vero CellsABSTRACT
Interferon-γ (IFN-γ) engenders strong antiproliferative responses, in part through activation of p53. However, the long-known IFN-γ-dependent upregulation of human Trp-tRNA synthetase (TrpRS), a cytoplasmic enzyme that activates tryptophan to form Trp-AMP in the first step of protein synthesis, is unexplained. Here we report a nuclear complex of TrpRS with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and with poly(ADP-ribose) polymerase 1 (PARP-1), the major PARP in human cells. The IFN-γ-dependent poly(ADP-ribosyl)ation of DNA-PKcs (which activates its kinase function) and concomitant activation of the tumor suppressor p53 were specifically prevented by Trp-SA, an analog of Trp-AMP that disrupted the TrpRS-DNA-PKcs-PARP-1 complex. The connection of TrpRS to p53 signaling in vivo was confirmed in a vertebrate system. These and further results suggest an unexpected evolutionary expansion of the protein synthesis apparatus to a nuclear role that links major signaling pathways.
Subject(s)
DNA-Activated Protein Kinase/metabolism , Interferon-gamma/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Signal Transduction/drug effects , Tryptophan-tRNA Ligase/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Catalytic Domain , Cell Culture Techniques , Cell Nucleus/drug effects , Cell Nucleus/enzymology , Cell Nucleus/metabolism , Cytoplasm/drug effects , Cytoplasm/enzymology , Cytoplasm/metabolism , DNA-Activated Protein Kinase/genetics , Electrophoresis, Polyacrylamide Gel , Embryo, Nonmammalian/enzymology , HeLa Cells , Humans , Immunoprecipitation , Interferon-gamma/physiology , Microscopy, Confocal , Models, Molecular , Phosphorylation , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/genetics , Protein Interaction Maps , Transfection , Tryptophan-tRNA Ligase/genetics , Tumor Suppressor Protein p53/genetics , Zebrafish/embryology , Zebrafish/metabolismABSTRACT
Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Antigens, CD/metabolism , Cadherins/metabolism , Catalytic Domain , Models, Molecular , Tryptophan/metabolism , Amino Acyl-tRNA Synthetases/genetics , Blotting, Western , Humans , Immunoprecipitation , Mutagenesis , Structure-Activity RelationshipABSTRACT
Aminoacyl tRNA synthetases are known for catalysis of aminoacylation. Significantly, some mammalian synthetases developed cytokine functions possibly linked to disease-causing mutations in tRNA synthetases. Not understood is how epitopes for cytokine signaling were introduced into catalytic scaffolds without disturbing aminoacylation. Here we investigate human tyrosyl-tRNA synthetase, where a catalytic-domain surface helix, next to the active site, was recruited for interleukin-8-like cytokine signaling. Taking advantage of our high resolution structure, the reciprocal impact of rational mutations designed to disrupt aminoacylation or cytokine signaling was investigated with multiple assays. The collective analysis demonstrated a protective fine-structure separation of aminoacylation from cytokine activities within the conserved catalytic domain. As a consequence, disease-causing mutations affecting cell signaling can arise without disturbing aminoacylation. These results with TyrRS also predict the previously unknown binding conformation of interleukin-8-like CXC cytokines.
Subject(s)
Cytokines/chemistry , Mutation , Tyrosine-tRNA Ligase/chemistry , Amino Acid Substitution , Aminoacylation , Biocatalysis , Catalytic Domain , Computer Simulation , Humans , Mutant Proteins/metabolism , Protein Binding , Tyrosine-tRNA Ligase/metabolismABSTRACT
In mammalian cells, specific aminoacyl-transfer RNA (tRNA) synthetases have cytokine functions that require interactions with partners outside of the translation apparatus. Little is known about these interactions and how they facilitate expanded functions that link protein translation to other cellular pathways. For example, an alternative splice fragment of tryptophanyl-tRNA synthetase (TrpRS) and a similar natural proteolytic fragment are potent angiostatic factors that act through the vascular endothelial-cadherin receptor and Akt signaling pathway. Here we demonstrate mobilization of TrpRS for exocytosis from endothelial cells and the potential for plasmin to activate the cytokine function of the extracellular synthetase. Direct physical evidence showed that the annexin II-S100A10 complex, which regulates exocytosis, forms a ternary complex with TrpRS. Functional studies demonstrate that both annexin II and S100A10 regulate trafficking of TrpRS. Thus, complexes of mammalian tRNA synthetases with seemingly disparate proteins may in general be relevant to understanding how their expanded functions are implemented.
Subject(s)
Angiostatic Proteins/metabolism , Annexin A2/metabolism , Cytokines/metabolism , Endothelial Cells/metabolism , Fibrinolysin/metabolism , S100 Proteins/metabolism , Tryptophan-tRNA Ligase/metabolism , Alternative Splicing/physiology , Angiostatic Proteins/genetics , Annexin A2/genetics , Cells, Cultured , Cytokines/genetics , Endothelial Cells/cytology , Exocytosis/physiology , Fibrinolysin/genetics , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Biosynthesis/physiology , Protein Transport/physiology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , S100 Proteins/genetics , Signal Transduction/physiology , Tryptophan-tRNA Ligase/geneticsABSTRACT
Among the various inhibitors known for enoyl-acyl carrier protein (ACP) reductases, triclosan and green tea catechins are two promising candidates. In the present study, we show, for the first time that epigallocatechin gallate (EGCG), a major component of green tea catechins, inhibits InhA, the enoyl-ACP reductase of Mycobacterium tuberculosis with an IC50 of 17.4muM. EGCG interferes with the binding of NADH to InhA. We also demonstrate that EGCG increased the inhibitory activity of triclosan towards InhA and vice versa. Direct binding assay using [(3)H]EGCG and fluorescence titration assay support the spectrophotometric/kinetic inhibition data. The biochemical data has been explained by docking simulation studies.
Subject(s)
Catechin/analogs & derivatives , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/antagonists & inhibitors , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/metabolism , Mycobacterium tuberculosis/enzymology , Triclosan/pharmacology , Catechin/chemistry , Catechin/pharmacology , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/chemistry , Enzyme Inhibitors/pharmacology , Models, Molecular , Molecular Structure , Protein Binding , Protein Structure, Tertiary , Spectrometry, Fluorescence , Triclosan/chemistryABSTRACT
Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.
Subject(s)
Cytokines/metabolism , Mutation , Tyrosine-tRNA Ligase/metabolism , Amino Acid Motifs/genetics , Amino Acid Substitution , Animals , Cattle , Cell Movement/drug effects , Cell Proliferation/drug effects , Chemotaxis, Leukocyte/drug effects , Chick Embryo , Endothelial Cells/cytology , Endothelial Cells/drug effects , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Mice , Mice, Nude , Models, Molecular , Neovascularization, Physiologic/drug effects , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Peptide Hydrolases/chemistry , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Protein Conformation , Protein Structure, Tertiary , Scattering, Small Angle , Tyrosine-tRNA Ligase/chemistry , Tyrosine-tRNA Ligase/genetics , X-Ray DiffractionABSTRACT
BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 A, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, alpha+beta ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein).
Subject(s)
Bacterial Proteins/chemistry , Coloring Agents/metabolism , Conserved Sequence , Heme/metabolism , Multienzyme Complexes/chemistry , Peroxidases/chemistry , Protein Folding , Amino Acid Motifs , Amino Acid Sequence , Bacterial Proteins/physiology , Bacteroides/enzymology , Catalytic Domain , Crystallography, X-Ray , Molecular Sequence Data , Multienzyme Complexes/physiology , Peroxidases/metabolism , Protein Binding , Protein Structure, Secondary , Shewanella/enzymologyABSTRACT
TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Coloring Agents/metabolism , Heme/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Peroxidases/chemistry , Peroxidases/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Molecular Sequence Data , Protein Binding , Shewanella/enzymologyABSTRACT
Higher eukaryote tRNA synthetases have expanded functions that come from enlarged, more differentiated structures that were adapted to fit aminoacylation function. How those adaptations affect catalytic mechanisms is not known. Presented here is the structure of a catalytically active natural splice variant of human tryptophanyl-tRNA synthetase (TrpRS) that is a potent angiostatic factor. This and related structures suggest that a eukaryote-specific N-terminal extension of the core enzyme changed substrate recognition by forming an active site cap. At the junction of the extension and core catalytic unit, an arginine is recruited to replace a missing landmark lysine almost 200 residues away. Mutagenesis, rapid kinetic, and substrate binding studies support the functional significance of the cap and arginine recruitment. Thus, the enzyme function of human TrpRS has switched more to the N terminus of the sequence. This switch has the effect of creating selective pressure to retain the N-terminal extension for functional expansion.