ABSTRACT
Assays drive drug discovery from the exploratory phases to the clinical testing of drug candidates. As such, numerous assay technologies and methodologies have arisen to support drug discovery efforts. Robust identification and characterization of tractable chemical matter requires biochemical, biophysical, and cellular approaches and often benefits from high-throughput methods. To increase throughput, efforts have been made to provide assays in miniaturized volumes which can be arrayed in microtiter plates to support the testing of as many as 100,000 samples/day. Alongside these efforts has been the growth of microtiter plate-free formats with encoded libraries that can support the screening of billions of compounds, a hunt for new drug modalities, as well as emphasis on more disease relevant formats using complex cell models of disease states. This review will focus on recent developments in high-throughput assay technologies applied to identify starting points for drug discovery. We also provide recommendations on strategies for implementing various assay types to select high quality leads for drug development.
Subject(s)
Drug Discovery/methods , Organic Chemicals/analysis , High-Throughput Screening Assays , Humans , Small Molecule Libraries/analysisABSTRACT
Nicotinamide phosphoribosyltransferase is a key metabolic enzyme that is a potential target for oncology. Utilizing publicly available crystal structures of NAMPT and in silico docking of our internal compound library, a NAMPT inhibitor, 1, obtained from a phenotypic screening effort was replaced with a more synthetically tractable scaffold. This compound then provided an excellent foundation for further optimization using crystallography driven structure based drug design. From this approach, two key motifs were identified, the (S,S) cyclopropyl carboxamide and the (S)-1-N-phenylethylamide that endowed compounds with excellent cell based potency. As exemplified by compound 27e such compounds could be useful tools to explore NAMPT biology in vivo.
Subject(s)
Amides/pharmacology , Cyclopropanes/pharmacology , Cytokines/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/pharmacology , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Adenosine/analogs & derivatives , Amides/chemical synthesis , Amides/chemistry , Crystallography, X-Ray , Cyclopropanes/chemical synthesis , Cyclopropanes/chemistry , Cytokines/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Molecular Docking Simulation , Molecular Structure , Nicotinamide Phosphoribosyltransferase/metabolism , Phenotype , Structure-Activity RelationshipABSTRACT
One of the central questions in the characterization of enzyme inhibitors is determining the mode of inhibition (MOI). Classically, this is done with a number of low-throughput methods in which inhibition models are fitted to the data. The ability to rapidly characterize the MOI for inhibitors arising from high-throughput screening in which hundreds to thousands of primary inhibitors may need to be characterized would greatly help in lead selection efforts. Here we describe a novel method for determining the MOI of a compound without the need for curve fitting of the enzyme inhibition data. We provide experimental data to demonstrate the utility of this new high-throughput MOI classification method based on nonparametric analysis of the activity derived from a small matrix of substrate and inhibitor concentrations (e.g., from a 4S × 4I matrix). Lists of inhibitors from four different enzyme assays are studied, and the results are compared with the previously described IC50-shift method for MOI classification. The MOI results from this method are in good agreement with the known MOI and compare favorably with those from the IC50-shift method. In addition, we discuss some advantages and limitations of the method and provide recommendations for utilization of this MOI classification method.
Subject(s)
Enzyme Inhibitors/chemistry , Enzymes/chemistry , High-Throughput Screening Assays/methods , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/classification , HumansABSTRACT
The thiazolidinediones (TZD) typified by rosiglitazone are the only approved therapeutics targeting PPARγ for the treatment of type-2 diabetes (T2DM). Unfortunately, despite robust insulin sensitizing properties, they are accompanied by a number of severe side effects including congestive heart failure, edema, weight gain, and osteoporosis. We recently identified PPARγ antagonists that bind reversibly with high affinity but do not induce transactivation of the receptor, yet they act as insulin sensitizers in mouse models of diabetes (SR1664).1 This Letter details our synthetic exploration around this novel series of PPARγ antagonists based on an N-biphenylmethylindole scaffold. Structure-activity relationship studies led to the identification of compound 46 as a high affinity PPARγ antagonist that exhibits antidiabetic properties following oral administration in diet-induced obese mice.
ABSTRACT
Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a "closed" to "open" conformation to accommodate the viral RNA template. Inhibitors that lock the "closed" or block the "open" conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a "closed" conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.
Subject(s)
Antiviral Agents/pharmacology , DNA-Directed RNA Polymerases/antagonists & inhibitors , Dengue Virus/drug effects , Dengue Virus/enzymology , Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Microbial Sensitivity Tests/methods , Chromatography, Liquid , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Dengue Virus/genetics , Dose-Response Relationship, Drug , Drug Discovery/methods , Drug Discovery/standards , Drug Evaluation, Preclinical/standards , High-Throughput Screening Assays/standards , Humans , Inhibitory Concentration 50 , Mass Spectrometry , Microbial Sensitivity Tests/standards , Reproducibility of Results , Small Molecule LibrariesABSTRACT
AMP-activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme with hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin, and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and ß subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the ß subunit and in the kinase domain of the α subunit, suggesting that the molecular binding site of the latter resides between the α and ß subunits. The distinct short- and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands.
Subject(s)
AMP-Activated Protein Kinases/chemistry , Allosteric Regulation , Biphenyl Compounds , Catalytic Domain , Deuterium Exchange Measurement , Enzyme Activation , Enzyme Activators/chemistry , Humans , Models, Molecular , Protein Binding , Protein Structure, Secondary , Pyrones/chemistry , Thiophenes/chemistryABSTRACT
The nuclear receptor (NR) superfamily is composed of 48 members in humans and includes receptors for steroid hormones, thyroid hormone, various lipids and oxysterols. This superfamily has been a rich source of drug targets for myriad diseases including inflammation, cancer, and metabolic disorders. Approximately half of the superfamily have well characterized natural ligands whereas the remaining receptors are considered orphan receptors and remain a focus of a number of investigators assessing their ability to be regulated by ligands. Here, we review recent discoveries that yield important insight into the druggability of three orphan nuclear receptors: the retinoic acid receptor-like orphan receptors (RORs), peroxisome proliferator-activated receptor γ (PPARγ), and liver receptor homolog-1 (LRH-1).
Subject(s)
Autoimmune Diseases/drug therapy , Metabolic Diseases/drug therapy , Orphan Nuclear Receptors/antagonists & inhibitors , Autoimmune Diseases/metabolism , Humans , Metabolic Diseases/metabolism , Orphan Nuclear Receptors/metabolism , PPAR gamma/agonists , PPAR gamma/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/pharmacology , Phosphatidylcholines/therapeutic use , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/antagonists & inhibitors , Receptors, Retinoic Acid/metabolism , Sterols/chemistry , Sterols/pharmacology , Sterols/therapeutic use , Thiazolidinediones/chemistry , Thiazolidinediones/pharmacology , Thiazolidinediones/therapeutic useABSTRACT
PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of antidiabetes drugs including rosiglitazone and pioglitazone. These drugs are full classical agonists for this nuclear receptor, but recent data have shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk5. Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent antidiabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of antidiabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ.
Subject(s)
Cyclin-Dependent Kinase 5/antagonists & inhibitors , Hypoglycemic Agents/pharmacology , PPAR gamma/metabolism , 3T3-L1 Cells , Adipocytes/drug effects , Adipocytes/metabolism , Adipose Tissue, White/drug effects , Adipose Tissue, White/metabolism , Animals , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Body Fluids/drug effects , COS Cells , Chlorocebus aethiops , Dietary Fats/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Hypoglycemic Agents/adverse effects , Hypoglycemic Agents/chemistry , Ligands , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Models, Molecular , Obesity/chemically induced , Obesity/metabolism , Osteogenesis/drug effects , PPAR gamma/agonists , PPAR gamma/chemistry , Phosphorylation/drug effects , Phosphoserine/metabolism , Rosiglitazone , Thiazolidinediones/adverse effects , Thiazolidinediones/pharmacology , Transcription, Genetic/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Weight Gain/drug effectsABSTRACT
Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.
Subject(s)
Phosphatidylcholines/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Bile Acids and Salts/biosynthesis , Bile Acids and Salts/metabolism , Bile Acids and Salts/pharmacology , Blood Glucose/metabolism , Cell Line , Disease Models, Animal , Fatty Liver/drug therapy , Fatty Liver/enzymology , HeLa Cells , Homeostasis/drug effects , Humans , Hypoglycemic Agents/pharmacology , Insulin Resistance/physiology , Ligands , Lipogenesis/drug effects , Liver/drug effects , Liver/enzymology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylcholines/pharmacology , Protein Binding , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/deficiency , Receptors, Cytoplasmic and Nuclear/genetics , Signal Transduction/drug effects , Triglycerides/metabolismABSTRACT
The vitamin D receptor (VDR) functions as an obligate heterodimer in complex with the retinoid X receptor (RXR). These nuclear receptors are multidomain proteins, and it is unclear how various domains interact with one another within the nuclear receptor heterodimer. Here, we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA-binding domains (DBDs), including the coactivator binding surfaces of both co-receptors, and that the sequence of the DNA response element can determine these dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that the ligand itself may play a role in DNA recognition. These data suggest a mechanism by which nuclear receptors show promoter specificity and have differential effects on various target genes, providing insight into the function of selective nuclear receptor modulators.
Subject(s)
Protein Interaction Domains and Motifs , Receptors, Calcitriol/chemistry , Retinoid X Receptors/chemistry , Alitretinoin , Binding Sites , Dihydroxycholecalciferols/chemistry , Humans , Ligands , Models, Molecular , Nuclear Receptor Coactivator 1/chemistry , Promoter Regions, Genetic , Protein Interaction Mapping , Protein Stability , Protein Structure, Tertiary , Receptors, Calcitriol/agonists , Receptors, Calcitriol/metabolism , Retinoid X Receptors/agonists , Retinoid X Receptors/metabolism , Tretinoin/chemistryABSTRACT
Retinoids are potent forms of vitamin A and are involved in a broad range of physiological processes and the pharmacological effects of retinoids are primarily mediated by the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Several natural and synthetic RAR modulators have proven to be clinically useful for a number of therapeutic indications including cancer, psoriasis, and diabetes. Unfortunately, these agents lead to a number of significant side effects. Most synthetic retinoid ligands are based on the retinoid scaffold and thus have similarities to the natural ligand with all previously disclosed RAR ligands having a carboxylic acid that makes a critical ionic bridge within the ligand binding domain of the receptors. The potential therapeutic value offered from RAR modulation provides the impetus to identify novel ligands based on unique scaffolds that may offer improved toxicity and pharmacokinetic profiles. Here we describe the identification of an atypical RAR inverse agonist that represents the first non-acid, non-retinoid direct modulator of RAR receptor subfamily. SR-0065 functions as a pan-RAR inverse agonist suppressing the basal activity of RARα, RARß, and RARγ, as well as inhibiting agonist-induced RAR activity. SR-0065 treatment enhanced receptor interaction with a peptide representative of the corepressor SMRT, and in cells SR-0065 enhances recruitment of SMRT to the promoter of the RARγ dependent gene, Cyp26A1. The acid form of SR-0065, SR-1758, was inactive in all assays. Thus, SR-0065 represents a new class of non-acid, non-retinoid RAR modulator that may be used as a point to initiate development of improved RAR-targeted drugs.
Subject(s)
Dioxanes/pharmacology , Quinolones/pharmacology , Receptors, Retinoic Acid/agonists , Retinoid X Receptors/agonists , Animals , Cell Line , Dioxanes/chemistry , Dose-Response Relationship, Drug , Humans , Ligands , Mice , Molecular Structure , Quinolones/chemistry , Structure-Activity RelationshipABSTRACT
Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule-receptor interactions, this technique has also been applied to study protein-protein complexes, such as mapping antibody-antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein-ligand interactions has had an impact on biology and drug discovery.
Subject(s)
Deuterium Exchange Measurement/methods , Mass Spectrometry/methods , Proteins/chemistry , Animals , Hormones/chemistry , Humans , Hydrogen/chemistry , Ligands , Models, Molecular , Protein Binding , Protein Conformation , Protein Kinases/chemistry , Protein Structure, Tertiary , Receptors, Cell Surface/chemistry , Receptors, Cytoplasmic and Nuclear/chemistryABSTRACT
The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339-345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity.
Subject(s)
Meclizine/pharmacology , Pyridines/pharmacology , Receptors, Cytoplasmic and Nuclear/metabolism , Recombinant Fusion Proteins/metabolism , Retinoid X Receptors/metabolism , Allosteric Site , Animals , Binding Sites , Constitutive Androstane Receptor , Humans , Mice , Models, Molecular , Nuclear Receptor Coactivator 1/chemistry , Protein Binding , Protein Multimerization , Protein Stability , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/chemistry , Recombinant Fusion Proteins/agonists , Recombinant Fusion Proteins/chemistry , Retinoid X Receptors/chemistry , Thermodynamics , Transcriptional ActivationABSTRACT
Peroxisome proliferator-activated receptor γ (PPARγ) is involved in expression of genes that control glucose and lipid metabolism. PPARγ is the molecular target of the thiazolidinedione (TZD) class of antidiabetic drugs. However, despite their clinical use these drugs are associated with numerous adverse effects, which are related to their full activation of PPARγ transcriptional responses. PPARγ partial agonists are the focus of development efforts towards second-generation PPARγ modulators with favorable pharmacology, potent insulin sensitization without the severe full agonists' adverse effects. In order to identify novel PPARγ partial agonist lead compounds, we developed a virtual screening protocol based on three-dimensional ligand-shape similarity and docking. Prioritization gave 235 compounds for experimental screening from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR)-a chemical library containing 340 000 compounds. Seven novel potent partial agonists were confirmed in cell-based transactivation and competitive binding assays. Our results illustrate a well-designed virtual screening campaign successfully identifying novel lead compounds as potential entry points for the development of antidiabetic drugs.
Subject(s)
Diabetes Mellitus/drug therapy , Hypoglycemic Agents , PPAR gamma/agonists , PPAR gamma/metabolism , Small Molecule Libraries , Animals , Binding Sites/drug effects , COS Cells , Chlorocebus aethiops , Diabetes Mellitus/physiopathology , High-Throughput Screening Assays , Hypoglycemic Agents/metabolism , Hypoglycemic Agents/pharmacology , Ligands , Models, Molecular , PPAR gamma/genetics , Protein Conformation/drug effects , Quantitative Structure-Activity Relationship , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Thiazolidinediones/metabolism , Thiazolidinediones/pharmacology , User-Computer InterfaceABSTRACT
Retinoic acid receptor-related orphan receptors (RORs) regulate a variety of physiological processes including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. Here we present the first high-affinity synthetic ligand for both RORalpha and RORgamma. In a screen against all 48 human nuclear receptors, the benzenesulfonamide liver X receptor (LXR) agonist N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317) inhibited transactivation activity of RORalpha and RORgamma but not RORbeta. T0901317 was found to directly bind to RORalpha and RORgamma with high affinity (K(i) = 132 and 51 nM, respectively), resulting in the modulation of the receptor's ability to interact with transcriptional cofactor proteins. T0901317 repressed RORalpha/gamma-dependent transactivation of ROR-responsive reporter genes and in HepG2 cells reduced recruitment of steroid receptor coactivator-2 by RORalpha at an endogenous ROR target gene (G6Pase). Using small interference RNA, we demonstrate that repression of the gluconeogenic enzyme glucose-6-phosphatase in HepG2 cells by T0901317 is ROR-dependent and is not due to the compound's LXR activity. In summary, T0901317 represents a novel chemical probe to examine RORalpha/gamma function and an excellent starting point for the development of ROR selective modulators. More importantly, our results demonstrate that small molecules can be used to target the RORs for therapeutic intervention in metabolic and immune disorders.
Subject(s)
Hydrocarbons, Fluorinated/pharmacology , Nuclear Receptor Subfamily 1, Group F, Member 1/agonists , Receptors, Retinoic Acid/agonists , Sulfonamides/pharmacology , Cell Line , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Nuclear Receptor Subfamily 1, Group F, Member 1/metabolism , Protein Binding/physiology , Receptors, Retinoic Acid/metabolism , Retinoic Acid Receptor gammaABSTRACT
Human carbonic anhydrase II (HCA II) is a monomeric zinc-containing metalloenzyme that catalyzes the hydration of CO(2) to form bicarbonate and a proton. The properties of the zinc have been extensively elucidated in catalysis but less well studied as a contributor to structure and stability. Apo-HCA II (without zinc) was prepared and compared to holo-HCA II: in crystallographic structural features, in backbone amide H/D exchange, and in thermal stability. The removal of zinc from the active site has no effect on either the topological fold of the enzyme or the ordered water network in the active site. However, the removal of the zinc alters the collective electrostatics of the apo-HCA II that result in the following differences from that of the holoenzyme: (1) the main thermal unfolding transition of the apo-HCA II is lowered by 8 degrees C, (2) the relative increase in thermal mobility of atoms of the apo-HCA II was not observed in the vicinity of the active site but manifested on the surface of the enzyme, and (3) the side chain of His 64, the proton shuttle residue that sits on the rim of the active site, is oriented outward and is associated with additional ordered "external" waters, as opposed to a near equal inward and outward orientation in the holo-HCA II.
Subject(s)
Apoproteins/chemistry , Carbonic Anhydrase II/chemistry , Solvents/chemistry , Zinc , Apoproteins/metabolism , Carbonic Anhydrase II/metabolism , Catalysis , Crystallography, X-Ray , Enzyme Stability , Holoenzymes/chemistry , Holoenzymes/metabolism , Humans , Static Electricity , Zinc/chemistry , Zinc/metabolismABSTRACT
Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Several members of the matrix metalloproteinase (MMP) family catalyze the hydrolysis of collagen triple helical structure. This study has utilized triple helical peptide (THP) substrates and inhibitors to dissect MMP-1 collagenolytic behavior. Analysis of MMP-1/THP interactions by hydrogen/deuterium exchange mass spectrometry followed by evaluation of wild type and mutant MMP-1 kinetics led to the identification of three noncatalytic regions in MMP-1 (residues 285-295, 302-316, and 437-457) and two specific residues (Ile-290 and Arg-291) that participate in collagenolysis. Ile-290 and Arg-291 contribute to recognition of triple helical structure and facilitate both the binding and catalysis of the triple helix. Evidence from this study and prior studies indicates that the MMP-1 catalytic and hemopexin-like domains collaborate in collagen catabolism by properly aligning the triple helix and coupling conformational states to facilitate hydrolysis. This study is the first to document the roles of specific residues within the MMP-1 hemopexin-like domain in substrate binding and turnover. Noncatalytic sites, such as those identified here, can ultimately be utilized to create THP inhibitors that target MMPs implicated in disease progression while sparing proteases with host-beneficial functions.
Subject(s)
Collagen/chemistry , Matrix Metalloproteinase 1/chemistry , Animals , Collagen/metabolism , Humans , Matrix Metalloproteinase 1/metabolism , Protein Structure, Secondary/physiology , Protein Structure, Tertiary/physiology , Substrate Specificity/physiologyABSTRACT
Here we describe an integrated software platform titled HD Desktop designed specifically to enhance the analysis of hydrogen/deuterium exchange (HDX) mass spectrometry data. HD Desktop integrates tools for data extraction with visualization components within a single web-based application. The interface design enables users to navigate from the peptide view to the sample and experiment levels, tracking all manipulations while updating the aggregate graphs in real time. HD Desktop is integrated with a relational database designed to provide performance enhancements, as well as a robust model for data storage and retrieval. Additional features of the software include retention time determination, which is achieved with the use of theoretical isotope fitting; here, we assume that the best theoretical fit will occur at the correct retention time for any given peptide. Peptide data consolidation for the rendering of data in 2D was realized by automating known and novel approaches. Designed to address broad needs of the HDX community, the platform presented here provides an efficient and manageable workflow for HDX data analysis and is freely available as a web tool at the project home page http://hdx.florida.scripps.edu.
Subject(s)
Deuterium Exchange Measurement , Information Storage and Retrieval/methods , Mass Spectrometry , Software , User-Computer Interface , Database Management Systems , Databases as Topic , Internet , Peptides/analysis , Proteins/analysisABSTRACT
The peroxisome proliferator-activated receptor is a member of the nuclear receptor superfamily of transcriptional regulators. Regulation of the nuclear receptors occurs through changes to the structure and dynamics of the ligand-binding domain. Therefore, the need has arisen for a rapid method capable of detecting changes in the dynamics of nuclear receptors following ligand binding. We recently described how solution-phase amide hydrogen/deuterium exchange (HDX) provides a biophysical technique for probing changes in protein dynamics induced by ligand interaction. Building from this platform, we have optimized the robustness of the differential HDX experiment by minimizing systematic errors, and have increased the efficiency of the chromatographic separation through the use of high-pressure liquid chromatography. Using knowledge gained previously from comprehensive HDX experiments of PPARgamma, a modest throughput method to probe changes in the dynamics of key regions of the receptor was developed. A collection of ten synthetic and endogenous PPARgamma ligands were characterized with this new method requiring approximately 24 h of analysis. This is a dramatic improvement over the 10 d of analysis that would have been required with our previous approach for comprehensive differential HDX analysis. In addition to demonstrating the utility of this approach, the study presented here is the first to measure changes to the dynamics of PPARgamma upon the binding of putative endogenous ligands.
Subject(s)
Deuterium/chemistry , Hydrogen/chemistry , Proteins/metabolism , Chromatography, High Pressure Liquid , Ligands , Mass Spectrometry , Protein BindingABSTRACT
Binding to helix 12 of the ligand-binding domain of PPARgamma is required for full agonist activity. Previously, the degree of stabilization of the activation function 2 (AF-2) surface was thought to correlate with the degree of agonism and transactivation. To examine this mechanism, we probed structural dynamics of PPARgamma with agonists that induced graded transcriptional responses. Here we present crystal structures and amide H/D exchange (HDX) kinetics for six of these complexes. Amide HDX revealed each ligand induced unique changes to the dynamics of the ligand-binding domain (LBD). Full agonists stabilized helix 12, whereas intermediate and partial agonists did not at all, and rather differentially stabilized other regions of the binding pocket. The gradient of PPARgamma transactivation cannot be accounted for solely through changes to the dynamics of AF-2. Thus, our understanding of allosteric signaling must be extended beyond the idea of a dynamic helix 12 acting as a molecular switch.