ABSTRACT
Monoclonal antibody (mAb) pharmaceuticals consist of a plethora of different proteoforms with different functional characteristics, including pharmacokinetics and pharmacodynamics, requiring their individual assessment. Current binding techniques do not distinguish between coexisting proteoforms requiring tedious production of enriched proteoforms. Here, we have developed an approach based on mobility shift-affinity capillary electrophoresis-mass spectrometry (ACE-MS), which permitted us to determine the binding of coexisting mAb proteoforms to Fc receptors (FcRs). For high-sensitivity MS analysis, we used a sheathless interface providing adequate mAb sensitivity allowing functional characterization of mAbs with a high sensitivity and dynamic range. As a model system, we focused on the interaction with the neonatal FcR (FcRn), which determines the half-life of mAbs. Depending on the oxidation status, proteoforms exhibited different electrophoretic mobility shifts in the presence of FcRn, which could be used to determine their affinity. We confirmed the decrease of the FcRn affinity with antibody oxidation and observed a minor glycosylation effect, with higher affinities for galactosylated glycoforms. Next to relative binding, the approach permits the determination of individual KD values in solution resulting in values of 422 and 139 nM for double-oxidized and non-oxidized variants. Hyphenation with native MS provides unique capabilities for simultaneous heterogeneity assessment for mAbs, FcRn, and complexes formed. The latter provides information on binding stoichiometry revealing 1:1 and 1:2 for antibody/FcRn complexes. The use of differently engineered Fc-only constructs allowed distinguishing between symmetric and asymmetric binding. The approach opens up unique possibilities for proteoform-resolved antibody binding studies to FcRn and can be extended to other FcRs and protein interactions.
Subject(s)
Electrophoresis, Capillary , Receptors, Fc , Antibodies, Monoclonal/metabolism , Glycosylation , Histocompatibility Antigens Class I/metabolism , Humans , Infant, Newborn , Mass Spectrometry , Receptors, Fc/metabolismABSTRACT
Removal of apoptotic cells by phagocytes (also called efferocytosis) is a crucial process for tissue homeostasis. Professional phagocytes express a plethora of surface receptors enabling them to sense and engulf apoptotic cells, thus avoiding persistence of dead cells and cellular debris and their consequent effects. Dysregulation of efferocytosis is thought to lead to secondary necrosis and associated inflammation and immune activation. Efferocytosis in primarily murine macrophages and dendritic cells has been shown to require TAM RTKs, with MERTK and AXL being critical for clearance of apoptotic cells. The functional role of human orthologs, especially the exact contribution of each individual receptor is less well studied. Here we show that human macrophages differentiated in vitro from iPSC-derived precursor cells express both AXL and MERTK and engulf apoptotic cells. TAM RTK agonism by the natural ligand growth-arrest specific 6 (GAS6) significantly enhanced such efferocytosis. Using a newly-developed mouse model of kinase-dead MERTK, we demonstrate that MERTK kinase activity is essential for efferocytosis in peritoneal macrophages in vivo. Moreover, human iPSC-derived macrophages treated in vitro with blocking antibodies or small molecule inhibitors recapitulated this observation. Hence, our results highlight a conserved MERTK function between mice and humans, and the critical role of its kinase activity in homeostatic efferocytosis.
Subject(s)
Macrophages/physiology , Phagocytosis/physiology , c-Mer Tyrosine Kinase/metabolism , Animals , Cell Differentiation , Cells, Cultured , HEK293 Cells , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/physiology , Intercellular Signaling Peptides and Proteins/physiology , Ligands , Macrophages/drug effects , Mice , Mice, Knockout , Phagocytosis/drug effects , Phagocytosis/genetics , Phosphatidylserines/pharmacology , c-Mer Tyrosine Kinase/agonists , c-Mer Tyrosine Kinase/geneticsABSTRACT
Metallo-ß-lactamases (MBLs) are an emerging cause of bacterial antibiotic resistance by hydrolysing all classes of ß-lactams except monobactams, and the MBLs are not inhibited by clinically available serine-ß-lactamase inhibitors. Two of the most commonly encountered MBLs in clinical isolates worldwide - the New Delhi metallo-ß-lactamase (NDM-1) and the Verona integron-encoded metallo-ß-lactamase (VIM-2) - are included in this study. A series of several NH-1,2,3-triazoles was prepared by a three-step protocol utilizing Banert cascade reaction as the key step. The inhibitor properties were evaluated in biochemical assays against the MBLs VIM-2, NDM-1 and GIM-1, and VIM-2 showed IC50 values down to nanomolar range. High-resolution crystal structures of four inhibitors in complex with VIM-2 revealed hydrogen bonds from the triazole inhibitors to Arg228 and to the backbone of Ala231 or Asn233, along with hydrophobic interactions to Trp87, Phe61 and Tyr67. The inhibitors show reduced MIC in synergy assays with Pseudomonas aeruginosa and Escherichia coli strains harbouring VIM enzymes. The obtained results will be useful for further structural guided design of MBL inhibitors.
Subject(s)
Triazoles/pharmacology , beta-Lactam Resistance/drug effects , beta-Lactamase Inhibitors/pharmacology , Anti-Bacterial Agents/pharmacology , Catalytic Domain , Crystallography, X-Ray , Drug Synergism , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , Klebsiella pneumoniae/drug effects , Meropenem/pharmacology , Molecular Structure , Protein Binding , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/metabolism , beta-Lactamase Inhibitors/chemical synthesis , beta-Lactamase Inhibitors/metabolism , beta-Lactamases/metabolismABSTRACT
Many class D ß-lactamases form dimers in solution. The functional basis of the dimerization of OXA-48-like class D ß-lactamases is not known, but in order to understand the structural requirements for dimerization of OXA-48, we have characterized the dimer interface. Size exclusion chromatography, small angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) were used to confirm the oligomeric state of OXA-48 in solution. X-ray crystallographic structures were used to elucidate the key interactions of dimerization. In silico residue scanning combined with site-directed mutagenesis was used to probe hot spots of dimerization. The affinity of dimerization was quantified using microscale thermophoresis, and the overall thermostability was investigated using differential scanning calorimetry. OXA-48 was consistently found to be a dimer in solution regardless of the method used, and the biological assembly found from the SAXS envelope is consistent with the dimer identified from the crystal structures. The buried chloride that interacts with Arg206 and Arg206' at the dimer interface was found to enhance the thermal stability by > 4 °C and crystal structures and mutations (R189A, R189A/R206A) identified several additional important ionic interactions. The affinity for OXA-48 R206A dimerization was in the picomolar range, thus revealing very high dimer affinity. In summary, OXA-48 has a very stable dimer interface, facilitated by noncovalent and predominantly charged interactions, which is stronger than the dimer interfaces previously described for other class D ß-lactamases. PDB CODES: The oxacillinase-48 (OXA-48) R206A structure has PDB ID: 5OFT and OXA-48 R189A has PDB ID: 6GOA.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Mutation , Protein Multimerization , beta-Lactamases/chemistry , beta-Lactamases/metabolism , Binding Sites , Crystallography, X-Ray , Escherichia coli Proteins/genetics , Models, Molecular , Mutagenesis, Site-Directed , Scattering, Small Angle , X-Ray Diffraction , beta-Lactamases/geneticsABSTRACT
Enzymatic depolymerization of recalcitrant polysaccharides plays a key role in accessing the renewable energy stored within lignocellulosic biomass, and natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. Here, a metagenome from a thermophilic microbial community was mined to yield a novel, thermostable cellulase, named mgCel6A, with activity on an industrial cellulosic substrate (sulfite-pulped Norway spruce) and a glucomannanase side activity. The enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) and a family 2 carbohydrate binding module (CBM2) that are connected by a linker rich in prolines and threonines. MgCel6A exhibited maximum activity at 85°C and pH 5.0 on carboxymethyl cellulose (CMC), but in prolonged incubations with the industrial substrate, the highest yields were obtained at 60°C, pH 6.0. Differential scanning calorimetry (DSC) indicated a Tm(app) of 76°C. Both functional data and the crystal structure, solved at 1.88 Å resolution, indicate that mgCel6A is an endoglucanase. Comparative studies with a truncated variant of the enzyme showed that the CBM increases substrate binding, while not affecting thermal stability. Importantly, at higher substrate concentrations the full-length enzyme was outperformed by the catalytic domain alone, underpinning previous suggestions that CBMs may be less useful in high-consistency bioprocessing.
Subject(s)
Cellulase/chemistry , Cellulase/metabolism , Cellulose/metabolism , Composting , Metagenome , Amino Acid Sequence , Catalytic Domain , Cellulase/genetics , Cloning, Molecular , Crystallography, X-Ray , Enzyme Stability , Hydrolysis , Kinetics , Protein Conformation , Sequence Homology , Substrate Specificity , TemperatureABSTRACT
ß-Lactam antibiotics are of utmost importance when treating bacterial infections in the medical community. However, currently their utility is threatened by the emergence and spread of ß-lactam resistance. The most prevalent resistance mechanism to ß-lactam antibiotics is expression of ß-lactamase enzymes. One way to overcome resistance caused by ß-lactamases, is the development of ß-lactamase inhibitors and today several ß-lactamase inhibitors e.g. avibactam, are approved in the clinic. Our focus is the oxacillinase-48 (OXA-48), an enzyme reported to spread rapidly across the world and commonly identified in Escherichia coli and Klebsiella pneumoniae. To guide inhibitor design, we used diversely substituted 3-aryl and 3-heteroaryl benzoic acids to probe the active site of OXA-48 for useful enzyme-inhibitor interactions. In the presented study, a focused fragment library containing 49 3-substituted benzoic acid derivatives were synthesised and biochemically characterized. Based on crystallographic data from 33 fragment-enzyme complexes, the fragments could be classified into R1 or R2 binders by their overall binding conformation in relation to the binding of the R1 and R2 side groups of imipenem. Moreover, binding interactions attractive for future inhibitor design were found and their usefulness explored by the rational design and evaluation of merged inhibitors from orthogonally binding fragments. The best inhibitors among the resulting 3,5-disubstituted benzoic acids showed inhibitory potential in the low micromolar range (IC50â¯=â¯2.9⯵M). For these inhibitors, the complex X-ray structures revealed non-covalent binding to Arg250, Arg214 and Tyr211 in the active site and the interactions observed with the mono-substituted fragments were also identified in the merged structures.
Subject(s)
Drug Design , Drug Resistance, Microbial/drug effects , Small Molecule Libraries/pharmacology , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Dose-Response Relationship, Drug , Escherichia coli/enzymology , Klebsiella pneumoniae/enzymology , Molecular Structure , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship , beta-Lactamase Inhibitors/chemical synthesis , beta-Lactamase Inhibitors/chemistryABSTRACT
Metallo-ß-lactamases (MBLs) threaten the effectiveness of ß-lactam antibiotics, including carbapenems, and are a concern for global public health. ß-Lactam/ß-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-ß-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC50) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC50 levels of the new thiol-based inhibitors were 0.66 µM (inhibitor 2a) and 0.62 µM (inhibitor 2b), and the equilibrium dissociation constant (KD ) of inhibitor 2a was 1.6 µM; thus, both were more potent inhibitors than l-captopril (IC50 = 47 µM; KD = 25 µM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than l-captopril.
Subject(s)
Achromobacter denitrificans , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , beta-Lactamases/genetics , Achromobacter denitrificans/drug effects , Achromobacter denitrificans/enzymology , Achromobacter denitrificans/genetics , Bacterial Proteins/metabolism , Captopril/pharmacology , Carbapenems/therapeutic use , Microbial Sensitivity Tests , Surface Plasmon Resonance , beta-Lactamase Inhibitors/therapeutic use , beta-Lactamases/metabolismABSTRACT
The increasing number of pathogens expressing metallo-ß-lactamases (MBLs), and in this way achieving resistance to ß-lactam antibiotics, is a significant threat to global public health. A promising strategy to treat such resistant pathogens is the co-administration of MBL inhibitors together with ß-lactam antibiotics. However, an MBL inhibitor suitable for clinical use has not yet been identified. Verona integron-encoded metallo-ß-lactamase 2 (VIM-2) is a widespread MBL with a broad substrate spectrum and hence is an interesting drug target for the treatment of ß-lactam-resistant infections. In this study, three triazolylthioacetamides were tested as inhibitors of VIM-2. One of the tested compounds showed clear inhibition of VIM-2, with an IC50 of 20â µM. The crystal structure of the inhibitor in complex with VIM-2 was obtained by DMSO-free co-crystallization and was solved at a resolution of 1.50â Å. To our knowledge, this is the first structure of a triazolylthioacetamide inhibitor in complex with an MBL. Analysis of the structure shows that the inhibitor binds to the two zinc ions in the active site of VIM-2 and revealed detailed information on the interactions involved. Furthermore, the crystal structure showed that binding of the inhibitor induced a conformational change of the conserved residue Trp87.
Subject(s)
Bacterial Proteins/chemistry , Pseudomonas aeruginosa/chemistry , Recombinant Fusion Proteins/chemistry , beta-Lactamase Inhibitors/chemistry , beta-Lactamases/chemistry , Amino Acid Sequence , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Inhibitory Concentration 50 , Models, Molecular , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Pseudomonas aeruginosa/enzymology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Thioacetamide/chemistry , Triazoles/chemistry , Tryptophan/chemistry , Tryptophan/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
The spread of antibiotic resistant bacteria is a global threat that shakes the foundations of modern healthcare. ß-Lactamases are enzymes that confer resistance to ß-lactam antibiotics in bacteria, and there is a critical need for new inhibitors of these enzymes for combination therapy together with an antibiotic. With this in mind, we have screened a library of 490 fragments to identify starting points for the development of new inhibitors of the class D ß-lactamase oxacillinase-48 (OXA-48) through surface plasmon resonance (SPR), dose-rate inhibition assays, and X-ray crystallography. Furthermore, we have uncovered structure-activity relationships and used alternate conformations from a crystallographic structure to grow a fragment into a more potent compound with a KD of 50 µM and an IC50 of 18 µM.
Subject(s)
Drug Design , Drug Resistance, Microbial/drug effects , Surface Plasmon Resonance , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Humans , Models, Molecular , Molecular Structure , Structure-Activity Relationship , beta-Lactamase Inhibitors/chemical synthesis , beta-Lactamase Inhibitors/chemistry , beta-Lactamases/geneticsABSTRACT
The inhibition of metallo-ß-lactamases (MBL) can prevent the hydrolysis of ß-lactam antibiotics and hence is a promising strategy for the treatment of antibiotic resistant infections. In this study, we present a novel reversible covalent inhibitor of the clinically relevant MBL New Delhi metallo-ß-lactamase 1 (NDM-1). Electrospray ionization-mass spectrometry (ESI-MS) and single site directed mutagenesis were used to show that the inhibitor forms a covalent bond with Lys224 in the active site of NDM-1. The inhibitor was further characterized using an enzyme inhibition assay, a surface plasmon resonance (SPR) based biosensor assay and covalent docking. The determined inhibition constant (KI(∗)) was 580nM and the inhibition constant for the initial complex (KI) was 76µM. To our knowledge, this inhibitor is the first example for a reversible covalent non-ß-lactam inhibitor targeting NDM-1 and a promising starting point for the design of potent covalent inhibitors.
Subject(s)
Drug Discovery , beta-Lactamase Inhibitors/chemical synthesis , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Captopril/chemistry , Captopril/pharmacology , Catalytic Domain , Enzyme Activation/drug effects , Kinetics , Meropenem , Models, Molecular , Mutagenesis, Site-Directed , Spectrometry, Mass, Electrospray Ionization , Surface Plasmon Resonance , Thienamycins/chemistry , Thienamycins/pharmacology , beta-Lactamase Inhibitors/chemistry , beta-Lactamases/chemistry , beta-Lactamases/geneticsABSTRACT
Metallo-ß-lactamases (MBLs) render bacteria resistant to ß-lactam antibiotics and are interesting drug targets to prevent the hydrolysis of ß-lactam antibiotics. So far, there are no MBL inhibitors in clinical use and particularly the design of broad spectrum inhibitors targeting several MBLs has been difficult. In this study, we report four fragments inhibiting the clinically relevant New Delhi metallo-ß-lactamase 1 (NDM-1) and Verona integron-encoded metallo-ß-lactamase 2 (VIM-2). The fragments were identified from a library using an orthogonal screening strategy combining a surface plasmon resonance (SPR) based assay and an enzyme inhibition assay. The identified fragments showed dissociation constants (KD) ranging from 181 to 2100 µM. The binding mode of the fragments was explored using QM-polarized ligand docking. All four fragments represent interesting scaffolds for the design of broad-spectrum MBL inhibitors.
Subject(s)
Drug Discovery , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Dose-Response Relationship, Drug , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Surface Plasmon Resonance , beta-Lactamase Inhibitors/chemical synthesis , beta-Lactamase Inhibitors/chemistryABSTRACT
Metallo-ß-lactamase (MBL) inhibitors can restore the function of carbapenem antibiotics and therefore help to treat infections of antibiotic resistant bacteria. In this study, we report novel fragments inhibiting the clinically relevant MBL Verona integron-encoded metallo-ß-lactamase (VIM-2). The fragments were identified from a library of 490 fragments using an orthogonal screening approach based on a surface plasmon resonance (SPR) based assay combined with an enzyme inhibition assay. The identified fragments showed IC50 values between 14 and 1500 µM and ligand efficiencies (LE) between 0.48 and 0.23 kcal/mol per heavy atom. For two of the identified fragments, crystal structures in complex with VIM-2 were obtained. The identified fragments represent novel inhibitor scaffolds and are good starting points for the design of potent MBL inhibitors. Furthermore, the established SPR based assay and the screening approach can be adapted to other MBLs and in this way improve the drug discovery process for this important class of drug targets.
Subject(s)
Pseudomonas aeruginosa/enzymology , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Carbapenems/pharmacology , Crystallography, X-Ray , Drug Design , Drug Resistance, Bacterial/drug effects , Humans , Models, Molecular , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Surface Plasmon ResonanceABSTRACT
The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose.
Subject(s)
Aquatic Organisms/chemistry , Peptide Hydrolases/metabolism , Protease Inhibitors/chemistry , Biological Assay/methods , Protease Inhibitors/pharmacology , Spectrum Analysis/methods , Surface Plasmon Resonance/methodsABSTRACT
A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric ß3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to ß3 GABA(A) receptors with micromolar affinity (K(D)<300 µM), thus extending previous evidence that ß3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with ß3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric ß3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for ß3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether ß3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site.
Subject(s)
Histamine Agents/pharmacology , Receptors, GABA-A/metabolism , Surface Plasmon Resonance/methods , Animals , Biosensing Techniques/methods , Cell Line , Histamine Agents/chemistry , Humans , Insecta , Protein Binding/physiology , Rats , Receptors, GABA-A/chemistryABSTRACT
A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (ß-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Surface Plasmon Resonance/methods , Alzheimer Disease/drug therapy , Alzheimer Disease/enzymology , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/isolation & purification , Animals , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/isolation & purification , Calcium/metabolism , Cell Line , Cloning, Molecular , Enzyme Inhibitors/chemistry , Enzymes, Immobilized/antagonists & inhibitors , Enzymes, Immobilized/genetics , Enzymes, Immobilized/isolation & purification , Enzymes, Immobilized/metabolism , Humans , Lipid Bilayers/metabolism , Models, MolecularABSTRACT
BACE-1 is one of the aspartic proteases involved in the cleavage of beta amyloid peptide, an initial step in the formation of amyloid plaques whose toxicity induces neuron death in Alzheimer's disease patients. One of the central issues in the search of novel BACE-1 inhibitors is the optimum pH for the binding of inhibitors to the enzyme. It is known that the enzyme has optimal catalytic activity at acidic pH, while cell active inhibitors may bind optimally at higher pH. In this work we determine the effect of the pH on the affinities of a set of inhibitors, with a variety of chemical motifs, for the ectodomain region of BACE-1 by a surface plasmon resonance (SPR) biosensor based assay. In order to understand the molecular interactions that underlie the diverse optimum pH for the binding of the various inhibitors as observed experimentally, we have calculated the titration curves for a set of BACE-1 ligand complexes. The results indicate that the pK(a) values of the titratable residues of the protein depend on the nature of the ligand involved, in disagreement with previous work. The enzyme-inhibitor structures with the resulting protonation states at pH values 4.5 and 7.4 served as the starting point for the prediction of the pH-dependent binding ranking. Our calculations reproduced the entire affinity ranking observed upon pH increase and most of the binding trends among inhibitors, especially at low pH. Finally, our cell-based assays indicate a possible correlation between high inhibitor affinity at both acidic and neutral pH values, with optimal cell response, a result that may open new venues for the search of potent BACE-1 inhibitors that are active at the cellular level.
Subject(s)
Enzyme Inhibitors/chemistry , Physical Phenomena , Amyloid beta-Protein Precursor , Enzyme Inhibitors/pharmacology , Humans , Ligands , Protease Nexins , Protein Structure, Tertiary , Receptors, Cell SurfaceABSTRACT
The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with K(D)=0.03 mM and a low-affinity site with K(D)=5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K(D)=5 microM at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca2+ is an efficient regulator under physiological conditions.
Subject(s)
Biosensing Techniques/methods , C-Reactive Protein/metabolism , Calcium/metabolism , Phosphorylcholine/metabolism , Surface Plasmon Resonance/methods , C-Reactive Protein/chemistry , Calcium/chemistry , Hydrogen-Ion Concentration , Kinetics , Phosphorylcholine/chemistry , Protein BindingABSTRACT
Increasing evidence indicates that polypeptide aggregation often involves a nucleation and a growth phase, although the relationship between the factors that determine these two phases has not yet been fully clarified. We present here an analysis of several mutations at different sites of the Abeta(1-40) peptide, including those associated with early onset forms of the Alzheimer's disease, which reveals that the effects of specific amino acid substitutions in the sequence of this peptide are strongly modulated by their structural context. Nevertheless, mutations at different positions perturb in a correlated manner the free energies of aggregation as well as the lag times and growth rates. We show that these observations can be rationalized in terms of the intrinsic propensities for aggregation of the Abeta(1-40) sequence, thus suggesting that, in the case of this peptide, the determinants of the thermodynamics and of the nucleation and growth of the aggregates have a similar physicochemical basis.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Peptide Fragments/chemistry , Thermodynamics , Humans , Kinetics , Models, Molecular , Protein Denaturation , Protein FoldingABSTRACT
Amyloid formation is a nucleation-dependent process that is accelerated dramatically in vivo and in vitro upon addition of appropriate fibril seeds. A potent species barrier can be effective in this reaction if donor and recipient come from different biological species. This species barrier is thought to reflect differences in the amino acid sequence between seed and target polypeptide. Here we present an in vitro mutagenic cross-seeding analysis of Alzheimer's Abeta(1-40) peptide in which we mapped out the effect of systematically varied amino acid replacements on the propensity of seed-dependent amyloid fibril formation. We find that the susceptibility of different peptides toward cross-seeding relates to the intrinsic aggregation propensity of the respective polypeptide chain and, therefore, to properties such as beta-sheet propensity and hydrophobicity. These data imply that the seed-dependent formation of amyloid-like fibrils is affected by the intrinsic properties of the polypeptide chain in a manner that is similar to what has been described previously for aggregation reactions in general. Hence, the nucleus acts in this case as a catalyst that promotes the fibrillation of different polypeptide chains according to their intrinsic structural predilection.