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1.
Drug Metab Dispos ; 51(10): 1419-1427, 2023 10.
Article in English | MEDLINE | ID: mdl-37429728

ABSTRACT

The metabolism of lufotrelvir, a novel phosphate prodrug of PF-00835231 for the treatment of COVID-19, was evaluated in healthy human volunteers and clinical trial participants with COVID-19 following intravenous infusion. The prodrug was completely converted to PF-00835231 that was subsequently cleared by hydrolysis, hydroxylation, ketoreduction, epimerization, renal clearance, and secretion into the feces. The main circulating metabolite was a hydrolysis product (M7) that was present at concentrations greater than PF-00835231, and this was consistent between healthy volunteers and participants with COVID-19. On administration of [14C]lufotrelvir, only 63% of the dose was obtained in excreta over 10 days and total drug-related material demonstrated a prolonged terminal phase half-life in plasma. A considerable portion of the labeled material was unextractable from fecal homogenate and plasma. The position of the carbon-14 atom in the labeled material was at a leucine carbonyl, and pronase digestion of the pellet derived from extraction of the fecal homogenate showed that [14C]leucine was released. SIGNIFICANCE STATEMENT: Lufotrelvir is an experimental phosphate prodrug intravenous therapy investigated for the potential treatment of COVID-19 in a hospital setting. The overall metabolism of lufotrelvir was determined in human healthy volunteers and clinical trial participants with COVID-19. Conversion of the phosphate prodrug to the active drug PF-00835231 was complete and the subsequent metabolic clearance of the active drug was largely via amide bond hydrolysis. Substantial drug-related material was not recovered due to loss of the carbon-14 label to endogenous metabolism.


Subject(s)
COVID-19 , Prodrugs , Humans , Carbon Radioisotopes/analysis , Infusions, Intravenous , RNA, Viral/analysis , Leucine , SARS-CoV-2 , Administration, Intravenous , Phosphates , Feces/chemistry
2.
Mol Cell ; 52(1): 135-145, 2013 Oct 10.
Article in English | MEDLINE | ID: mdl-24119403

ABSTRACT

The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.


Subject(s)
Bacterial Proteins/metabolism , CRISPR-Associated Proteins/metabolism , RNA, Bacterial/metabolism , Ribonucleases/metabolism , Thermus thermophilus/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , CRISPR-Associated Proteins/chemistry , CRISPR-Associated Proteins/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , High-Throughput Nucleotide Sequencing , Microscopy, Electron , Models, Molecular , Protein Conformation , Protein Subunits , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , Ribonucleases/chemistry , Ribonucleases/genetics , Sequence Analysis, RNA , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship , Thermus thermophilus/genetics
3.
Br J Clin Pharmacol ; 85(10): 2332-2340, 2019 10.
Article in English | MEDLINE | ID: mdl-31269280

ABSTRACT

AIMS: Drug disposition in children may vary from adults due to age-related variation in drug metabolism. Microdose studies present an innovation to study pharmacokinetics (PK) in paediatrics; however, they should be used only when the PK is dose linear. We aimed to assess dose linearity of a [14 C]midazolam microdose, by comparing the PK of an intravenous (IV) microtracer (a microdose given simultaneously with a therapeutic midazolam dose), with the PK of a single isolated microdose. METHODS: Preterm to 2-year-old infants admitted to the intensive care unit received [14 C]midazolam IV as a microtracer or microdose, followed by dense blood sampling up to 36 hours. Plasma concentrations of [14 C]midazolam and [14 C]1-hydroxy-midazolam were determined by accelerator mass spectrometry. Noncompartmental PK analysis was performed and a population PK model was developed. RESULTS: Of 15 infants (median gestational age 39.4 [range 23.9-41.4] weeks, postnatal age 11.4 [0.6-49.1] weeks), 6 received a microtracer and 9 a microdose of [14 C]midazolam (111 Bq kg-1 ; 37.6 ng kg-1 ). In a 2-compartment PK model, bodyweight was the most significant covariate for volume of distribution. There was no statistically significant difference in any PK parameter between the microdose and microtracer, nor in the area under curve ratio [14 C]1-OH-midazolam/[14 C]midazolam, showing the PK of midazolam to be linear within the range of the therapeutic and microdoses. CONCLUSION: Our data support the dose linearity of the PK of an IV [14 C]midazolam microdose in children. Hence, a [14 C]midazolam microdosing approach may be used as an alternative to a therapeutic dose of midazolam to study developmental changes in hepatic CYP3A activity in young children.


Subject(s)
Hypnotics and Sedatives/administration & dosage , Midazolam/administration & dosage , Models, Biological , Administration, Intravenous , Age Factors , Area Under Curve , Carbon Radioisotopes , Dose-Response Relationship, Drug , Humans , Hypnotics and Sedatives/pharmacokinetics , Infant , Infant, Newborn , Intensive Care Units , Midazolam/analogs & derivatives , Midazolam/pharmacokinetics , Tissue Distribution
4.
Br J Clin Pharmacol ; 80(1): 157-67, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25619398

ABSTRACT

AIMS: The aims of the study were to compare [(14)C]-paracetamol ([(14)C]-PARA) paediatric pharmacokinetics (PK) after administration mixed in a therapeutic dose or an isolated microdose and to develop further and validate accelerator mass spectrometry (AMS) bioanalysis in the 0-2 year old age group. METHODS: [(14)C]-PARA concentrations in 10-15 µl plasma samples were measured after enteral or i.v. administration of a single [(14)C]-PARA microdose or mixed in with therapeutic dose in infants receiving PARA as part of their therapeutic regimen. RESULTS: Thirty-four infants were included in the PARA PK analysis for this study: oral microdose (n = 4), i.v. microdose (n = 6), oral therapeutic (n = 6) and i.v. therapeutic (n = 18). The respective mean clearance (CL) values (SDs in parentheses) for these dosed groups were 1.46 (1.00) l h(-1), 1.76 (1.07) l h(-1), 2.93 (2.08) l h(-1) and 2.72 (3.10) l h(-1), t(1/2) values 2.65 h, 2.55 h, 8.36 h and 7.16 h and dose normalized AUC(0-t) (mg l(-1) h) values were 0.90 (0.43), 0.84 (0.57), 0.7 (0.79) and 0.54 (0.26). CONCLUSIONS: All necessary ethical, scientific, clinical and regulatory procedures were put in place to conduct PK studies using enteral and systemic microdosing in two European centres. The pharmacokinetics of a therapeutic dose (mg kg(-1)) and a microdose (ng kg(-1)) in babies between 35 to 127 weeks post-menstrual age. [(14)C]-PARA pharmacokinetic parameters were within a two-fold range after a therapeutic dose or a microdose. Exploratory studies using doses significantly less than therapeutic doses may offer ethical and safety advantages with increased bionalytical sensitivity in selected exploratory paediatric pharmacokinetic studies.


Subject(s)
Acetaminophen/administration & dosage , Acetaminophen/pharmacokinetics , Carbon Radioisotopes , Acetaminophen/blood , Administration, Intravenous , Administration, Oral , Dose-Response Relationship, Drug , Female , Humans , Infant , Infant, Newborn , Male , Mass Spectrometry
5.
J Biol Chem ; 288(36): 26235-26245, 2013 Sep 06.
Article in English | MEDLINE | ID: mdl-23864660

ABSTRACT

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a dimeric flavoprotein that catalyzes the NADH- and oxygen-dependent para-hydroxylation of 3-hydroxybenzoate to 2,5-dihydroxybenzoate. In this study, we report the crystal structure of 3HB6H as expressed in Escherichia coli. The overall fold of 3HB6H is similar to that of p-hydroxybenzoate hydroxylase and other flavoprotein aromatic hydroxylases. Unexpectedly, a lipid ligand is bound to each 3HB6H monomer. Mass spectral analysis identified the ligand as a mixture of phosphatidylglycerol and phosphatidylethanolamine. The fatty acid chains occupy hydrophobic channels that deeply penetrate into the interior of the substrate-binding domain of each subunit, whereas the hydrophilic part is exposed on the protein surface, connecting the dimerization domains via a few interactions. Most remarkably, the terminal part of a phospholipid acyl chain is directly involved in the substrate-binding site. Co-crystallized chloride ion and the crystal structure of the H213S variant with bound 3-hydroxybenzoate provide hints about oxygen activation and substrate hydroxylation. Essential roles are played by His-213 in catalysis and Tyr-105 in substrate binding. This phospholipid-assisted strategy to control regioselective aromatic hydroxylation is of relevance for optimization of flavin-dependent biocatalysts.


Subject(s)
Bacterial Proteins/chemistry , Flavoproteins/chemistry , Mixed Function Oxygenases/chemistry , Phospholipids/chemistry , Rhodococcus/enzymology , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Flavoproteins/genetics , Flavoproteins/metabolism , Gene Expression , Gentisates/chemistry , Gentisates/metabolism , Hydroxylation , Mass Spectrometry , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Mutation, Missense , NAD/chemistry , NAD/genetics , NAD/metabolism , Phospholipids/genetics , Phospholipids/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodococcus/genetics
6.
Anal Chem ; 86(15): 7635-41, 2014 Aug 05.
Article in English | MEDLINE | ID: mdl-25033319

ABSTRACT

The increasing role of accelerator mass spectrometry (AMS) in biomedical research necessitates modernization of the traditional sample handling process. AMS was originally developed and used for carbon dating, therefore focusing on a very high precision but with a comparably low sample throughput. Here, we describe the combination of automated sample combustion with an elemental analyzer (EA) online coupled to an AMS via a dedicated interface. This setup allows direct radiocarbon measurements for over 70 samples daily by AMS. No sample processing is required apart from the pipetting of the sample into a tin foil cup, which is placed in the carousel of the EA. In our system, up to 200 AMS analyses are performed automatically without the need for manual interventions. We present results on the direct total (14)C count measurements in <2 µL human plasma samples. The method shows linearity over a range of 0.65-821 mBq/mL, with a lower limit of quantification of 0.65 mBq/mL (corresponding to 0.67 amol for acetaminophen). At these extremely low levels of activity, it becomes important to quantify plasma specific carbon percentages. This carbon percentage is automatically generated upon combustion of a sample on the EA. Apparent advantages of the present approach include complete omission of sample preparation (reduced hands-on time) and fully automated sample analysis. These improvements clearly stimulate the standard incorporation of microtracer research in the drug development process. In combination with the particularly low sample volumes required and extreme sensitivity, AMS strongly improves its position as a bioanalysis method.


Subject(s)
Automation , Mass Spectrometry/methods , Limit of Detection
7.
Anal Bioanal Chem ; 406(5): 1437-46, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24357008

ABSTRACT

The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions ('spikes') on their surface and are unique in having either T = 3 or T = 4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either near the spike apices (historically the 'α-determinant') or in the 'floor' regions between them (the 'ß-determinant'). Native mass spectrometry (MS) and gas-phase electrophoretic mobility molecular analysis (GEMMA) were used to monitor the titration of HBV capsids with the antigen-binding domain (Fab) of mAb 3120, which has long defined the ß-determinant. Both methods readily distinguished Fab binding to the two capsid morphologies and could provide accurate masses and dimensions for these large immune complexes, which range up to ~8 MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryo-electron microscopy analysis for preliminary characterisation of virus-antibody complexes.


Subject(s)
Antigen-Antibody Complex/chemistry , Antigens, Viral/chemistry , Capsid/chemistry , Hepatitis B virus/chemistry , Immunoglobulin Fab Fragments/chemistry , Antigens, Viral/immunology , Binding Sites , Capsid/immunology , Hepatitis B virus/immunology , Humans , Mass Spectrometry/methods , Protein Binding , Protein Interaction Domains and Motifs , Spectrometry, Mass, Electrospray Ionization/methods
8.
Mol Cell Proteomics ; 11(11): 1430-41, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22918228

ABSTRACT

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Inverted Repeat Sequences/genetics , Multiprotein Complexes/metabolism , Pseudomonas aeruginosa/metabolism , Tandem Mass Spectrometry/methods , Escherichia coli/genetics , Models, Molecular , Multiprotein Complexes/chemistry , Protein Binding , Protein Stability , Protein Subunits/chemistry , Protein Subunits/metabolism , Pseudomonas aeruginosa/genetics
9.
Proc Natl Acad Sci U S A ; 108(25): 10092-7, 2011 Jun 21.
Article in English | MEDLINE | ID: mdl-21536913

ABSTRACT

Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a "seed sequence" at the 5' end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.


Subject(s)
Base Sequence , Escherichia coli , Macromolecular Substances/metabolism , Pseudomonas aeruginosa , RNA/genetics , RNA/metabolism , Adaptive Immunity/genetics , Adaptive Immunity/immunology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Endoribonucleases/genetics , Endoribonucleases/metabolism , Escherichia coli/genetics , Escherichia coli/immunology , Macromolecular Substances/chemistry , Models, Molecular , Molecular Sequence Data , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/immunology
10.
J Am Chem Soc ; 135(17): 6504-12, 2013 May 01.
Article in English | MEDLINE | ID: mdl-23597076

ABSTRACT

Infection of humans by hepatitis B virus (HBV) induces the copious production of antibodies directed against the capsid protein (Cp). A large variety of anticapsid antibodies have been identified that differ in their epitopes. These data, and the status of the capsid as a major clinical antigen, motivate studies to achieve a more detailed understanding of their interactions. In this study, we focused on the Fab fragments of two monoclonal antibodies, E1 and 3120. E1 has been shown to bind to the side of outward-protruding spikes whereas 3120 binds to the "floor" region of the capsid, between spikes. We used hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the effects on HBV capsids of binding these antibodies. Conventionally, capsids loaded with saturating amounts of Fabs would be too massive to be readily amenable to HDX-MS. However, by focusing on the Cp protein, we were able to acquire deuterium uptake profiles covering the entire 149-residue sequence and reveal, in localized detail, changes in H/D exchange rates accompanying antibody binding. We find increased protection of the known E1 and 3120 epitopes on the capsid upon binding and show that regions distant from the epitopes are also affected. In particular, the α2a helix (residues 24-34) and the mobile C-terminus (residues 141-149) become substantially less solvent-exposed. Our data indicate that even at substoichiometric antibody binding an overall increase in the rigidity of the capsid is elicited, as well as a general dampening of its breathing motions.


Subject(s)
Capsid Proteins/immunology , Hepatitis B Antibodies/immunology , Hepatitis B virus/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Deuterium , Epitopes , Hepatitis B Core Antigens/immunology , Humans , Hydrogen , Immunoglobulin Fab Fragments/immunology , Mass Spectrometry , Models, Molecular , Protein Conformation
11.
Sci Adv ; 9(50): eadj9394, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-38100587

ABSTRACT

The Night Watch, one of the most famous masterpieces by Rembrandt, is the subject of a large research and conservation project. For the conservation treatment, it is of great importance to understand its current condition. Correlated nano-tomography using x-ray fluorescence and ptychography revealed a-so far unknown-lead-containing "layer", which likely acts as a protective impregnation layer applied on the canvas before the quartz-clay ground was applied. This layer might explain the presence of lead soap protrusions in areas where no other lead components are present. In addition to the three-dimensional elemental mapping, ptychography visualizes and quantifies components not detectable by hard x-ray fluorescence such as the organic fraction and quartz. The first-time use of this combination of synchrotron-based techniques on a historic paint micro-sample shows it to be an important tool to better interpret the results of noninvasive imaging techniques operating on the macroscale.

12.
J Struct Biol ; 177(2): 273-82, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22266117

ABSTRACT

Expression of the protruding (P) domain of the norovirus capsid protein, in vitro, results in the formation of P dimers and larger oligomers, 12-mer and 24-mer P particles. All these P complexes retain the authentic antigenicity and carbohydrate-binding function of the norovirus capsid. They have been used as tools to study norovirus-host interactions, and the 24-mer P particle has been proposed as a vaccine and vaccine platform against norovirus and other pathogens. In view of their pharmaceutical interest it is important to characterise the structure, stability and dynamics of these protein complexes. Here we use a native mass spectrometric approach. We analyse the P particles under both non-reducing and reducing conditions, as it is known that the macromolecular assemblies are stabilised by inter-subunit disulphide bonding. A novel 18-mer complex is identified, and we show that under reducing conditions the 24-mer dissociates into P dimers that reassemble into the 12-mer small P particle and another novel 36-mer complex. The collisional cross-sections of the 12-mer and 24-mer P particles determined by ion mobility MS are in good agreement with theoretical predictions and electron microscopy data. We propose a model structure for the 18-mer based on ion mobility experiments. Our results demonstrate the interchangeable nature and dynamic relationship of all P domain complexes and confirm their binding activity to the host receptors - human histo blood group antigens (HBGAs). These findings, together with the identification of the 18-mer and 36-mer P complexes add new information to the intriguing interactions of the norovirus P domain.


Subject(s)
Capsid Proteins/chemistry , Norovirus/physiology , Blood Group Antigens/chemistry , Dithiothreitol/chemistry , Host-Pathogen Interactions , Humans , Hydrogen-Ion Concentration , Molecular Weight , Multiprotein Complexes/chemistry , Protein Binding , Protein Stability , Protein Structure, Quaternary , Reducing Agents/chemistry , Saliva/chemistry , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry
13.
Anal Chem ; 84(16): 7227-32, 2012 Aug 21.
Article in English | MEDLINE | ID: mdl-22882109

ABSTRACT

Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.


Subject(s)
Antibodies/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Antibodies/isolation & purification , CHO Cells , Chromatography, Ion Exchange , Cricetinae , Cricetulus , Immunoglobulin G/analysis , Immunoglobulin G/isolation & purification
14.
Chembiochem ; 13(6): 829-36, 2012 Apr 16.
Article in English | MEDLINE | ID: mdl-22416028

ABSTRACT

Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35-375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin-binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post-selection characterisation, through the use of complementary approaches as described in this study.


Subject(s)
Aptamers, Nucleotide/chemistry , Streptavidin/chemistry , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/metabolism , Base Sequence , Binding Sites , Kinetics , Ligands , Oligonucleotides/chemistry , Streptavidin/genetics , Streptavidin/metabolism
15.
Mol Cell Proteomics ; 9(8): 1742-51, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20418222

ABSTRACT

Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP1(60) (3.36-MDa) and VP1(80) (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1(180)). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1(180) particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP1(60) and VP1(80) species.


Subject(s)
Norwalk virus/physiology , Spectrometry, Mass, Electrospray Ionization/methods , Virus Assembly/physiology , Capsid/chemistry , Capsid/ultrastructure , Microscopy, Atomic Force , Norwalk virus/ultrastructure , Osmolar Concentration , Particle Size
16.
Angew Chem Int Ed Engl ; 51(52): 12992-6, 2012 Dec 21.
Article in English | MEDLINE | ID: mdl-23172610

ABSTRACT

Antibody profiling: native mass spectrometry analysis of intact antibodies can be achieved with improved speed, sensitivity, and mass resolution by using a modified orbitrap instrument. Complex mixtures of monoclonal antibodies can be resolved and their glycan "fingerprints" can be profiled. Noncovalent interactions are maintained, thus allowing antibody-antigen binding to be measured.


Subject(s)
Antibodies, Monoclonal/chemistry , Mass Spectrometry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antigens/immunology , Glycosylation , HEK293 Cells , Humans , Interleukin-6/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology
17.
Clin Pharmacokinet ; 61(5): 697-707, 2022 05.
Article in English | MEDLINE | ID: mdl-34997559

ABSTRACT

BACKGROUND AND OBJECTIVE: In microdose studies, drug pharmacokinetics is measured in humans after administration of subtherapeutic doses. While previous microdose studies focused primarily on plasma pharmacokinetics, we set out to evaluate the feasibility of microdosing for a pharmacokinetic assessment in subcutaneous tissue and epithelial lining fluid. METHODS: Healthy subjects received a single intravenous bolus injection of a microdose of [14C]ciprofloxacin (1.1 µg, 7 kBq) with (cohort A, n = 9) or without (cohort B, n = 9) a prior intravenous infusion of a therapeutic dose of unlabeled ciprofloxacin (400 mg). Microdialysis and bronchoalveolar lavage were applied for determination of subcutaneous and intrapulmonary drug concentrations. Microdose [14C]ciprofloxacin was quantified by accelerator mass spectrometry and therapeutic-dose ciprofloxacin by liquid chromatography-tandem mass spectrometry. RESULTS: The pharmacokinetics of therapeutic-dose ciprofloxacin (cohort A) in plasma, subcutaneous tissue, and epithelial lining fluid was in accordance with previous data. In plasma and subcutaneous tissue, the dose-adjusted area under the concentration-time curve of microdose ciprofloxacin was similar in cohorts A and B and within an 0.8-fold to 1.1-fold range of the area under the concentration-time curve of therapeutic-dose ciprofloxacin. Penetration of microdose ciprofloxacin into subcutaneous tissue was similar in cohorts A and B and comparable to that of therapeutic-dose ciprofloxacin with subcutaneous tissue-to-plasma area under the concentration-time curve ratios of 0.44, 0.44, and 0.38, respectively. Penetration of microdose ciprofloxacin into epithelial lining fluid was highly variable and failed to predict the epithelial lining fluid penetration of therapeutic-dose ciprofloxacin. CONCLUSIONS: Our study confirms the feasibility of microdosing for pharmacokinetic measurements in plasma and subcutaneous tissue. Microdosing combined with microdialysis is a potentially useful tool in clinical antimicrobial drug development, but its applicability for the assessment of pulmonary pharmacokinetics with bronchoalveolar lavage requires further studies. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03177720 (registered 6 June, 2017).


Subject(s)
Anti-Bacterial Agents , Ciprofloxacin , Area Under Curve , Dose-Response Relationship, Drug , Feasibility Studies , Humans , Pharmaceutical Preparations
18.
Biochemistry ; 50(19): 4209-17, 2011 May 17.
Article in English | MEDLINE | ID: mdl-21486042

ABSTRACT

Putrescine oxidase (PuO) from Rhodococcus erythropolis is a soluble homodimeric flavoprotein, which oxidizes small aliphatic diamines. In this study, we report the crystal structures and cofactor binding properties of wild-type and mutant enzymes. From a structural viewpoint, PuO closely resembles the sequence-related human monoamine oxidases A and B. This similarity is striking in the flavin-binding site even if PuO does not covalently bind the cofactor as do the monoamine oxidases. A remarkable conserved feature is the cis peptide conformation of the Tyr residue whose conformation is important for substrate recognition in the active site cavity. The structure of PuO in complex with the reaction product reveals that Glu324 is crucial in recognizing the terminal amino group of the diamine substrate and explains the narrow substrate specificity of the enzyme. The structural analysis also provides clues for identification of residues that are responsible for the competitive binding of ADP versus FAD (~50% of wild-type PuO monomers isolated are occupied by ADP instead of FAD). By replacing Pro15, which is part of the dinucleotide-binding domain, enzyme preparations were obtained that are almost 100% in the FAD-bound form. Furthermore, mutants have been designed and prepared that form a covalent 8α-S-cysteinyl-FAD linkage. These data provide new insights into the molecular basis for substrate recognition in amine oxidases and demonstrate that engineering of flavoenzymes to introduce covalent linkage with the cofactor is a possible route to develop more stable protein molecules, better suited for biocatalytic purposes.


Subject(s)
Coenzymes/chemistry , Coenzymes/metabolism , Flavin-Adenine Dinucleotide/chemistry , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Rhodococcus/enzymology , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Biocatalysis , Coenzymes/genetics , Crystallography, X-Ray , Flavin-Adenine Dinucleotide/metabolism , Humans , Models, Molecular , Monoamine Oxidase/chemistry , Monoamine Oxidase/metabolism , Mutagenesis, Site-Directed , Oxidoreductases Acting on CH-NH Group Donors/genetics , Protein Binding/genetics , Protein Conformation , Protein Multimerization/genetics , Rhodococcus/genetics , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship
19.
Transgenic Res ; 20(5): 1033-42, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21188635

ABSTRACT

ER resident glycoproteins, including ectopically expressed recombinant glycoproteins, carry so-called high-mannose type N-glycans, which can be at different stages of processing. The presence of heterogeneous high-mannose type glycans on ER-retained therapeutic proteins is undesirable for specific therapeutic applications. Previously, we described an Arabidopsis alg3-2 glycosylation mutant in which aberrant Man(5)GlcNAc(2) mannose type N-glycans are transferred to proteins. Here we show that the alg3-2 mutation reduces the N-glycan heterogeneity on ER resident glycoproteins in seeds. We compared the properties of a scFv-Fc, with a KDEL ER retention tag (MBP10) that was expressed in seeds of wild type and alg3-2 plants. N-glycans on these antibodies from mutant seeds were predominantly of the intermediate Man(5)GlcNAc(2) compared to Man(8)GlcNAc(2) and Man(7)GlcNAc(2) isoforms on MBP10 from wild-type seeds. The presence of aberrant N-glycans on MBP10 did not seem to affect MBP10 dimerisation nor binding of MBP10 to its antigen. In alg3-2 the fraction of underglycosylated MBP10 protein forms was higher than in wild type. Interestingly, the expression of MBP10 resulted also in underglycosylation of other, endogenous glycoproteins.


Subject(s)
Arabidopsis/metabolism , Plants, Genetically Modified/metabolism , Polysaccharides/metabolism , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/immunology , Arabidopsis/genetics , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Glycosylation , Maltose-Binding Proteins/genetics , Maltose-Binding Proteins/immunology , Maltose-Binding Proteins/metabolism , Mannose/genetics , Mannose/metabolism , Mutation , Plants, Genetically Modified/genetics , Polysaccharides/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Seeds/genetics , Seeds/metabolism , Single-Chain Antibodies/genetics
20.
Chem Soc Rev ; 39(5): 1633-55, 2010 May.
Article in English | MEDLINE | ID: mdl-20419213

ABSTRACT

Traditionally, mass spectrometry has been a powerful analytical method enabling the structural analysis of small molecules, and later on peptides and proteins. With the advent of native mass spectrometry, using a combination of electrospray ionisation and time of flight analysis, mass spectrometry could also be applied to the mass determination of large protein complexes such as ribosomes and whole viruses. More recently, ion mobility has been coupled to mass spectrometry providing a new dimension in the analysis of biomolecules, with ion mobility separating ions according to differences in size and shape. In the context of native mass spectrometry, ion mobility mass spectrometry opens up avenues for the detailed structural analysis of large and heterogeneous protein complexes, providing information on the stoichiometry, topology and cross section of these assemblies and their composite subunits. With these characteristics, ion mobility mass spectrometry offers a complementary tool in the context of structural biology. Here, we critically review the development, instrumentation, approaches and applications of ion mobility in combination with mass spectrometry, focusing on the analysis of larger proteins and protein assemblies (185 references).


Subject(s)
Ions/chemistry , Mass Spectrometry , Proteins/chemistry , Models, Molecular , Protein Conformation , Protein Folding
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