ABSTRACT
Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral ß-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open ß-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.
Subject(s)
Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Endocytosis/drug effects , HEK293 Cells , Humans , Models, Molecular , Neostriatum/cytology , Neurons/drug effects , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Structure-Activity RelationshipABSTRACT
The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A2AR-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson's disease.
Subject(s)
Adenosine A2 Receptor Antagonists/chemistry , Allosteric Site , Parkinson Disease/drug therapy , Receptor, Adenosine A2A/chemistry , Adenosine A2 Receptor Antagonists/metabolism , Adenosine A2 Receptor Antagonists/therapeutic use , Animals , Antiparkinson Agents/chemistry , Antiparkinson Agents/metabolism , Antiparkinson Agents/therapeutic use , Crystallography, X-Ray , Humans , Ligands , Protein Structure, Tertiary , Receptor, Adenosine A2A/metabolism , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/metabolism , Sf9 Cells , Spodoptera , Triazines/chemistry , Triazines/metabolism , Triazoles/chemistry , Triazoles/metabolism , Tyrosine/chemistry , Tyrosine/metabolismABSTRACT
The impact of structural biology on drug discovery is well documented, and the workhorse technique for the past 30 years or so has been X-ray crystallography. With the advent of several technological improvements, including direct electron detectors, automation, better microscope vacuums and lenses, phase plates and improvements in computing power enabled by GPUs, it is now possible to record and analyse images of protein structures containing high-resolution information. This review, from a pharmaceutical perspective, highlights some of the most relevant and interesting protein structures for the pharmaceutical industry and shows examples of how ligand-binding sites, membrane proteins, both big and small, pseudo symmetry and complexes are being addressed by this technique.
Subject(s)
Cryoelectron Microscopy/methods , Drug Discovery , Crystallography, X-RayABSTRACT
Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE.
Subject(s)
Anti-Asthmatic Agents/pharmacology , Immunoglobulin E/metabolism , Models, Molecular , Omalizumab/pharmacology , Receptors, IgE/antagonists & inhibitors , Allosteric Site , Amino Acid Substitution , Crystallography, X-Ray , Fluorescence Resonance Energy Transfer , Humans , Immunoglobulin E/chemistry , Immunoglobulin E/genetics , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Fab Fragments/pharmacology , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin Fc Fragments/pharmacology , Omalizumab/chemistry , Omalizumab/genetics , Omalizumab/metabolism , Pliability , Point Mutation , Protein Conformation , Protein Interaction Domains and Motifs , Protein Refolding , Receptors, IgE/chemistry , Receptors, IgE/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Solubility , Surface Plasmon ResonanceABSTRACT
We describe the identification and characterization of a series of covalent inhibitors of the C-terminal kinase domain (CTKD) of MSK1. The initial hit was identified via a high-throughput screening and represents a rare example of a covalent inhibitor which acts via an SNAr reaction of a 2,5-dichloropyrimidine with a cysteine residue (Cys440). The covalent mechanism of action was supported by in vitro biochemical experiments and was confirmed by mass spectrometry. Ultimately, the displacement of the 2-chloro moiety was confirmed by crystallization of an inhibitor with the CTKD. We also disclose the crystal structures of three compounds from this series bound to the CTKD of MSK1, in addition to the crystal structures of two unrelated RSK2 covalent inhibitors bound to the CTKD of MSK1.
ABSTRACT
Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.
Subject(s)
Cryoelectron Microscopy/methods , Cycloparaffins/chemistry , Escherichia coli Proteins/isolation & purification , Escherichia coli/physiology , Multidrug Resistance-Associated Proteins/isolation & purification , Polymers/chemistry , Cryoelectron Microscopy/instrumentationABSTRACT
Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.
Subject(s)
GTP-Binding Protein alpha Subunits, Gs/chemistry , Receptors, Dopamine D1/agonists , Receptors, Dopamine D1/chemistry , Binding Sites , Crystallography, X-Ray , Humans , In Vitro Techniques , Ligands , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Engineering , Protein Structure, Quaternary , Recombinant Proteins/chemistryABSTRACT
Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors.
Subject(s)
Protein Multimerization/drug effects , Receptors, Tumor Necrosis Factor, Type I/chemistry , Signal Transduction/drug effects , Small Molecule Libraries/pharmacology , Tumor Necrosis Factor-alpha/chemistry , Algorithms , Animals , Binding, Competitive/drug effects , Humans , Models, Molecular , Protein Binding/drug effects , Protein Conformation/drug effects , Receptors, Tumor Necrosis Factor, Type I/metabolism , Small Molecule Libraries/chemistry , Tumor Necrosis Factor-alpha/metabolismABSTRACT
We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.
Subject(s)
Antibodies, Monoclonal/metabolism , Multiprotein Complexes/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Small Molecule Libraries/metabolism , Tumor Necrosis Factor-alpha/metabolism , Antibodies, Monoclonal/pharmacology , Cells, Cultured , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/metabolism , HEK293 Cells , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Protein Binding/drug effects , Protein Conformation/drug effects , Receptors, Tumor Necrosis Factor, Type I/chemistry , Signal Transduction/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Tumor Necrosis Factor-alpha/chemistryABSTRACT
Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by FcεRI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and FcεRI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with FcεRI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.
Subject(s)
Antibodies, Anti-Idiotypic/metabolism , Crystallization/methods , Immunoglobulin E/metabolism , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Fc Fragments/metabolism , Omalizumab/metabolism , Antibodies, Anti-Idiotypic/chemistry , Crystallography, X-Ray/methods , Humans , Immunoglobulin E/chemistry , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fc Fragments/chemistry , Omalizumab/pharmacology , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn's disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Arthritis, Experimental/drug therapy , Protein Multimerization/drug effects , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/immunology , Cell Line , Crystallography, X-Ray , Drug Discovery , Male , Mice , Molecular Dynamics Simulation , Neutrophil Infiltration/drug effects , Neutrophils/drug effects , Neutrophils/immunology , Protein Stability/drug effects , Protein Structure, Quaternary/drug effects , Receptors, Tumor Necrosis Factor, Type I/immunology , Receptors, Tumor Necrosis Factor, Type I/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Signal Transduction/immunology , Structure-Activity Relationship , Treatment Outcome , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/isolation & purification , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/ultrastructureABSTRACT
The SAR and pharmacokinetic profiles of a series of multi-isoform PI3K inhibitors based on a 3,4-dihydro-2H-benzo[1,4]oxazine scaffold are disclosed.
Subject(s)
Benzoxazines/chemistry , Chemistry, Pharmaceutical/methods , Oxazines/chemical synthesis , Oxazines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Area Under Curve , Benzoxazines/pharmacology , Crystallography, X-Ray/methods , Drug Design , Humans , Inflammation , Inhibitory Concentration 50 , Models, Chemical , Molecular Structure , Protein Isoforms , Structure-Activity RelationshipABSTRACT
The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.
Subject(s)
Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/pharmacokinetics , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Piperazines/pharmacology , Piperazines/pharmacokinetics , Piperidines/pharmacology , Transplantation, Homologous , Administration, Oral , Animals , Biological Availability , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/metabolism , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/pharmacokinetics , Immunosuppressive Agents/pharmacology , Mice , Molecular Docking Simulation , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Piperazines/administration & dosage , Piperazines/metabolism , Piperidines/administration & dosage , Piperidines/metabolism , Protein ConformationABSTRACT
Therapeutic and diagnostic applications of monoclonal antibodies often require careful selection of binders that recognize specific epitopes on the target molecule to exert a desired modulation of biological function. Here we present a proof-of-concept application for the rational design of an epitope-specific antibody binding with the target protein Keap1, by grafting pre-defined structural interaction patterns from the native binding partner protein, Nrf2, onto geometrically matched positions of a set of antibody scaffolds. The designed antibodies bind to Keap1 and block the Keap1-Nrf2 interaction in an epitope-specific way. One resulting antibody is further optimised to achieve low-nanomolar binding affinity by in silico redesign of the CDRH3 sequences. An X-ray co-crystal structure of one resulting design reveals that the actual binding orientation and interface with Keap1 is very close to the design model, despite an unexpected CDRH3 tilt and VH/VL interface deviation, which indicates that the modelling precision may be improved by taking into account simultaneous CDR loops conformation and VH/VL orientation optimisation upon antibody sequence change. Our study confirms that, given a pre-existing crystal structure of the target protein-protein interaction, hotspots grafting with CDR loop swapping is an attractive route to the rational design of an antibody targeting a pre-selected epitope.
Subject(s)
Antibodies, Monoclonal/immunology , Epitopes/chemistry , Kelch-Like ECH-Associated Protein 1/chemistry , NF-E2-Related Factor 2/chemistry , Amino Acid Sequence/genetics , Antibodies, Monoclonal/chemistry , Antibody Affinity/immunology , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/immunology , Computer Simulation , Crystallography, X-Ray , Epitopes/immunology , Humans , Kelch-Like ECH-Associated Protein 1/genetics , Kelch-Like ECH-Associated Protein 1/immunology , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/immunology , Protein Binding/genetics , Protein Binding/immunologyABSTRACT
Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9-2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism.
Subject(s)
DNA, Single-Stranded/chemistry , DNA, Viral/chemistry , Exodeoxyribonucleases/chemistry , Oligonucleotides/chemistry , Siphoviridae/chemistry , Viral Proteins/chemistry , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Gene Expression , Hydrogen Bonding , Models, Molecular , Mutagenesis, Site-Directed , Oligonucleotides/metabolism , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Siphoviridae/enzymology , Structure-Activity Relationship , Substrate Specificity , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.
Subject(s)
Immunoglobulin Fab Fragments/blood , Immunoglobulin Variable Region/blood , Serum Albumin/immunology , Animals , Antibody Affinity , Half-Life , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , MiceABSTRACT
G-protein coupled receptors (GPCRs) are the targets of over half of all prescribed drugs today. The UniProt database has records for about 800 proteins classified as GPCRs, but drugs have only been developed against 50 of these. Thus, there is huge potential in terms of the number of targets for new therapies to be designed. Several breakthroughs in GPCRs biased pharmacology, structural biology, modelling and scoring have resulted in a resurgence of interest in GPCRs as drug targets. Therefore, an international conference, sponsored by the Royal Society, with world-renowned researchers from industry and academia was recently held to discuss recent progress and highlight key areas of future research needed to accelerate GPCR drug discovery. Several key points emerged. Firstly, structures for all three major classes of GPCRs have now been solved and there is increasing coverage across the GPCR phylogenetic tree. This is likely to be substantially enhanced with data from x-ray free electron sources as they move beyond proof of concept. Secondly, the concept of biased signalling or functional selectivity is likely to be prevalent in many GPCRs, and this presents exciting new opportunities for selectivity and the control of side effects, especially when combined with increasing data regarding allosteric modulation. Thirdly, there will almost certainly be some GPCRs that will remain difficult targets because they exhibit complex ligand dependencies and have many metastable states rendering them difficult to resolve by crystallographic methods. Subtle effects within the packing of the transmembrane helices are likely to mask and contribute to this aspect, which may play a role in species dependent behaviour. This is particularly important because it has ramifications for how we interpret pre-clinical data. In summary, collaborative efforts between industry and academia have delivered significant progress in terms of structure and understanding of GPCRs and will be essential for resolving problems associated with the more difficult targets in the future.
Subject(s)
Drug Discovery , Receptors, G-Protein-Coupled/metabolism , Animals , Computer Simulation , Cooperative Behavior , Crystallography , Drug Industry , Humans , Models, Molecular , UniversitiesABSTRACT
An X-ray crystal structure of Kelch-like ECH-associated protein (Keap1) co-crystallised with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)-1 a) was obtained. This X-ray crystal structure provides breakthrough experimental evidence for the true binding mode of the hit compound (S,R,S)-1 a, as the ligand orientation was found to differ from that of the initial docking model, which was available at the start of the project. Crystallographic elucidation of this binding mode helped to focus and drive the drug design process more effectively and efficiently.