ABSTRACT
Neuronopathic glycosphingolipidoses are a sub-group of lysosomal storage disorders for which there are presently no effective therapies. Here, we evaluated the potential of substrate reduction therapy (SRT) using an inhibitor of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide (GL1) and related glycosphingolipids. The substrates that accumulate in Sandhoff disease (e.g., ganglioside GM2 and its nonacylated derivative, lyso-GM2) are distal to the drug target, GCS. Treatment of Sandhoff mice with a GCS inhibitor that has demonstrated CNS access (Genz-682452) reduced the accumulation of GL1 and GM2, as well as a variety of disease-associated substrates in the liver and brain. Concomitant with these effects was a significant decrease in the expression of CD68 and glycoprotein non-metastatic melanoma B protein (Gpnmb) in the brain, indicating a reduction in microgliosis in the treated mice. Moreover, using in vivo imaging, we showed that the monocytic biomarker translocator protein (TSPO), which was elevated in Sandhoff mice, was normalized following Genz-682452 treatment. These positive effects translated in turn into a delay (â¼28 days) in loss of motor function and coordination, as measured by rotarod latency, and a significant increase in longevity (â¼17.5%). Together, these results support the development of SRT for the treatment of gangliosidoses, particularly in patients with residual enzyme activity.
Subject(s)
Carbamates/pharmacology , Enzyme Inhibitors/pharmacology , Glucosyltransferases/antagonists & inhibitors , Quinuclidines/pharmacology , Sandhoff Disease/enzymology , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Disease Models, Animal , Female , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Ligands , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , Mass Spectrometry , Mice , Mice, Knockout , Molecular Imaging , Receptors, GABA/metabolism , Sandhoff Disease/diagnosis , Sandhoff Disease/genetics , Sandhoff Disease/therapy , Sphingolipids/metabolism , beta-Hexosaminidase beta Chain/genetics , beta-Hexosaminidase beta Chain/metabolismABSTRACT
SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure-activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.
Subject(s)
Amino Acid Transport Systems, Neutral/antagonists & inhibitors , Biological Assay/methods , Amino Acid Transport Systems, Neutral/metabolism , Animals , Cell Line , Fluorescence , Humans , Isotope Labeling , Membrane Potentials , Xenopus laevisABSTRACT
Certain recessively inherited diseases result from an enzyme deficiency within lysosomes. In mucopolysaccharidoses (MPS), a defect in glycosaminoglycan (GAG) degradation leads to GAG accumulation followed by progressive organ and multiple system dysfunctions. Current methods of GAG analysis used to diagnose and monitor the diseases lack sensitivity and throughput. Here we report a LC-MS method with accurate metabolite mass analysis for identifying and quantifying biomarkers for MPS type I without the need for extensive sample preparation. The method revealed 225 LC-MS features that were >1000-fold enriched in urine, plasma and tissue extracts from untreated MPS I mice compared to MPS I mice treated with iduronidase to correct the disorder. Levels of several trisaccharides were elevated >10000-fold. To validate the clinical relevance of our method, we confirmed the presence of these biomarkers in urine, plasma and cerebrospinal fluid from MPS I patients and assessed changes in their levels after treatment.
Subject(s)
Biomarkers/blood , Biomarkers/urine , Mucopolysaccharidosis I/blood , Mucopolysaccharidosis I/urine , Animals , Chromatography, Liquid , Disease Models, Animal , Female , Glycosaminoglycans/blood , Heparitin Sulfate/blood , Humans , Iduronidase/blood , Male , Mice , Trisaccharides/bloodABSTRACT
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ABSTRACT
Cytochrome P450 monooxygenases CYP101A1 and MycG catalyze regio- and stereospecific oxidations of their respective substrates, d-camphor and mycinamicin IV. Despite the low sequence homology between the two enzymes (29% identity) and differences in size and hydrophobicity of their substrates, the conformational changes that occur upon substrate binding in both enzymes as determined by solution NMR methods show some striking similarities. Many of the same secondary structural features in both enzymes are perturbed, suggesting the existence of a common mechanism for substrate binding and recognition in the P450 superfamily.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Camphor 5-Monooxygenase/chemistry , Camphor 5-Monooxygenase/metabolism , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Camphor/chemistry , Camphor/metabolism , Catalytic Domain , Macrolides/chemistry , Macrolides/metabolism , Magnetic Resonance Spectroscopy , Protein Conformation , Stereoisomerism , Substrate SpecificityABSTRACT
MycG is a P450 monooxygenase that catalyzes the sequential hydroxylation and epoxidation of mycinamicin IV (M-IV), the last two steps in the biosynthesis of mycinamicin II, a macrolide antibiotic isolated from Micromonospora griseorubida. The crystal structure of MycG with M-IV bound was previously determined but showed the bound substrate in an orientation that did not rationalize the observed regiochemistry of M-IV hydroxylation. Nuclear magnetic resonance paramagnetic relaxation enhancements provided evidence of an orientation of M-IV in the MycG active site more compatible with the observed chemistry, but substrate-induced changes in the enzyme structure were not characterized. We now describe the use of amide 1H-15N residual dipolar couplings as experimental restraints in solvated "soft annealing" molecular dynamics simulations to generate solution structural ensembles of M-IV-bound MycG. Chemical shift perturbations, hydrogen-deuterium exchange, and 15N relaxation behavior provide insight into the dynamic and electronic perturbations in the MycG structure in response to M-IV binding. The solution and crystallographic structures are compared, and the possibility that the crystallographic orientation of bound M-IV represents an inhibitory mode is discussed.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Molecular Dynamics Simulation , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Solutions , Substrate SpecificityABSTRACT
Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H2O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450(cam) (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures. Mutations were made to test the involvement of the observed conformational changes in substrate binding and recognition. All of the mutations performed based upon the NMR-detected perturbations, even those remote from the active site, resulted in modified substrate selectivity, enzyme efficiency and/or haem iron spin state. The results demonstrate that solution NMR can provide insights into enzyme structure-function relationships that are difficult to obtain by other methods.
Subject(s)
Cytochrome P-450 Enzyme System/chemistry , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Binding Sites , Camphor 5-Monooxygenase/chemistry , Camphor 5-Monooxygenase/genetics , Camphor 5-Monooxygenase/metabolism , Catalytic Domain , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Mutation , Protein Binding , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ~4 Å of the oxygen of iron-ligated water.