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1.
PLoS Negl Trop Dis ; 13(5): e0007373, 2019 05.
Article in English | MEDLINE | ID: mdl-31120889

ABSTRACT

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Antiprotozoal Agents/administration & dosage , Benzodiazepines/administration & dosage , Pyrroles/administration & dosage , Trypanosomiasis, African/drug therapy , Animals , Antibodies, Monoclonal/chemistry , Antiprotozoal Agents/chemistry , Benzodiazepines/chemistry , Female , Humans , Mice , Mice, Inbred BALB C , Pyrroles/chemistry , Trypanosoma brucei brucei/drug effects , Trypanosomiasis, African/parasitology
2.
Drug Discov Today Technol ; 23: 83-90, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28647091

ABSTRACT

The majority of antibody therapeutics have been isolated from target-led drug discovery, where many years of target research preceded drug program initiation. However, as the search for validated targets becomes more challenging and target space becomes increasingly competitive, alternative strategies, such as phenotypic drug discovery, are gaining favour. This review highlights successful examples of antibody phenotypic screens that have led to clinical drug candidates. We also review the requirements for performing an effective antibody phenotypic screen, including antibody enrichment and target identification strategies. Finally, the future impact of phenotypic drug discovery on antibody drug pipelines will be discussed.


Subject(s)
Antibodies/chemistry , Drug Discovery , Drug Evaluation, Preclinical , Humans
3.
Oncotarget ; 7(42): 68278-68291, 2016 10 18.
Article in English | MEDLINE | ID: mdl-27626702

ABSTRACT

Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.


Subject(s)
Immunotherapy/methods , Neoplasms/therapy , Receptors, Tumor Necrosis Factor, Type II/metabolism , T-Lymphocytes, Regulatory/metabolism , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Drug Screening Assays, Antitumor/methods , Female , HEK293 Cells , Humans , Jurkat Cells , Mice, Inbred BALB C , NF-kappa B/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/therapy , Phenotype , Receptors, Tumor Necrosis Factor, Type II/agonists , Receptors, Tumor Necrosis Factor, Type II/genetics , Signal Transduction/drug effects , T-Lymphocytes, Regulatory/drug effects
4.
Mol Cancer ; 14: 147, 2015 Jul 31.
Article in English | MEDLINE | ID: mdl-26227951

ABSTRACT

BACKGROUND: Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets. METHODS: ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin. RESULTS: Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model. CONCLUSIONS: Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.


Subject(s)
Antineoplastic Agents/pharmacology , Biological Products/pharmacology , Drug Discovery/methods , Drug Screening Assays, Antitumor/methods , Animals , Antigens, CD/immunology , Antigens, CD/metabolism , Antigens, Neoplasm , Apoptosis/drug effects , Biomarkers, Tumor , Carcinoma, Non-Small-Cell Lung , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/immunology , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Surface Display Techniques , Cisplatin/pharmacology , Disease Models, Animal , Humans , Lung Neoplasms , Mice , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Peptide Library , Phenotype , Single-Chain Antibodies/pharmacology , Spheroids, Cellular , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
5.
Chem Biol ; 22(1): 117-28, 2015 Jan 22.
Article in English | MEDLINE | ID: mdl-25544043

ABSTRACT

Describing, understanding, and modulating the function of the cell require elucidation of the structures of macromolecular assemblies. Here, we describe an integrative method for modeling heteromeric complexes using as a starting point disassembly pathways determined by native mass spectrometry (MS). In this method, the pathway data and other available information are encoded as a scoring function on the positions of the subunits of the complex. The method was assessed on its ability to reproduce the native contacts in five benchmark cases with simulated MS data and two cases with real MS data. To illustrate the power of our method, we purified the yeast initiation factor 3 (eIF3) complex and characterized it by native MS and chemical crosslinking MS. We established substoichiometric binding of eIF5 and derived a model for the five-subunit eIF3 complex, at domain level, consistent with its role as a scaffold for other initiation factors.


Subject(s)
Eukaryotic Initiation Factor-3/analysis , Models, Molecular , Peptide Initiation Factors/analysis , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/metabolism , Tandem Mass Spectrometry , Eukaryotic Initiation Factor-3/metabolism , Peptide Initiation Factors/metabolism , Protein Binding , ROC Curve , Saccharomyces cerevisiae Proteins/metabolism
6.
J Biol Chem ; 284(37): 25404-11, 2009 Sep 11.
Article in English | MEDLINE | ID: mdl-19592493

ABSTRACT

Toll-like receptors (TLRs) mediate responses to pathogen-associated molecules as part of the vertebrate innate immune response to infection. Receptor dimerization is coupled to downstream signal transduction by the recruitment of a post-receptor complex containing the adaptor protein MyD88 and the IRAK protein kinases. In this work, we show that the death domains of human MyD88 and IRAK-4 assemble into closed complexes having unusual stoichiometries of 7:4 and 8:4, the Myddosome. Formation of the Myddosome is likely to be a key event for TLR4 signaling in vivo as we show here that pathway activation requires that the receptors cluster into lipid rafts. Taken together, these findings indicate that TLR activation causes the formation of a highly oligomeric signaling platform analogous to the death-inducing signaling complex of the Fas receptor pathway.


Subject(s)
Interleukin-1 Receptor-Associated Kinases/chemistry , Myeloid Differentiation Factor 88/chemistry , Cell Line , Cross-Linking Reagents/pharmacology , Gene Expression Regulation , Humans , Mass Spectrometry/methods , Membrane Microdomains/chemistry , Models, Biological , Protein Structure, Tertiary , Scattering, Radiation , Signal Transduction , Ultracentrifugation , X-Rays , fas Receptor/metabolism
7.
Proteins ; 75(2): 360-72, 2009 May 01.
Article in English | MEDLINE | ID: mdl-18831052

ABSTRACT

The human AU RNA binding protein/enoyl-Coenzyme A hydratase (AUH) is a 3-hydroxy-3-methylglutaconyl-CoA dehydratase in the leucine degradation pathway. It also possesses an RNA-binding activity to AUUU repeats, which involves no known conserved RNA-binding domains and is seemingly unrelated to the enzymatic activity. In this study, we performed mass spectrometric analyses to elucidate the oligomeric states of AUH in the presence and absence of RNA. With a short RNA (AUUU) or without RNA, AUH mainly exists as a trimer in solution. On the other hand, the AUH trimer dimerizes upon binding to one molecule of a long RNA containing 24 repeats of the AUUU motif, (AUUU)(24)A. AUH was crystallized with the long RNA. Although the RNA was disordered in the crystalline lattice, the AUH structure was determined as an asymmetric dimer of trimers with a kink in the alignment of the trimer axes, resulting in the formation of two clefts with significantly different sizes.


Subject(s)
Enoyl-CoA Hydratase/chemistry , Enoyl-CoA Hydratase/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA/chemistry , RNA/metabolism , Crystallization , Humans , Mass Spectrometry , Protein Binding , Protein Conformation , Protein Multimerization , Protein Structure, Quaternary , Ultracentrifugation
8.
Proc Natl Acad Sci U S A ; 105(47): 18139-44, 2008 Nov 25.
Article in English | MEDLINE | ID: mdl-18599441

ABSTRACT

The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.


Subject(s)
Eukaryotic Initiation Factor-3/chemistry , Tandem Mass Spectrometry/methods , HeLa Cells , Humans , Models, Molecular
9.
Nat Protoc ; 3(7): 1139-52, 2008.
Article in English | MEDLINE | ID: mdl-18600219

ABSTRACT

Here we describe a detailed protocol for both data collection and interpretation with respect to ion mobility-mass spectrometry analysis of large protein assemblies. Ion mobility is a technique that can separate gaseous ions based on their size and shape. Specifically, within this protocol, we cover general approaches to data interpretation, methods of predicting whether specific model structures for a given protein assembly can be separated by ion mobility, and generalized strategies for data normalization and modeling. The protocol also covers basic instrument settings and best practices for both observation and detection of large noncovalent protein complexes by ion mobility-mass spectrometry.


Subject(s)
Mass Spectrometry/methods , Models, Molecular , Multiprotein Complexes/chemistry , Data Interpretation, Statistical , Ion Transport , Models, Statistical
10.
J Biol Chem ; 283(21): 14629-35, 2008 May 23.
Article in English | MEDLINE | ID: mdl-18347020

ABSTRACT

The Drosophila Toll receptor, which functions in both embryonic patterning and innate immunity to fungi and Gram-positive bacteria, is activated by a dimeric cytokine ligand, Spätzle (Spz). Previous studies have suggested that one Spz cross-links two Toll receptor molecules to form an activated complex. Here we report electron microscopy structures of the Toll ectodomain in the absence and presence of Spz. Contrary to expectations, Spz does not directly cross-link two Toll ectodomains. Instead, Spz binding at the N-terminal end of Toll predominantly induces the formation of a 2:2 complex, with two sites of interaction between the ectodomain chains, one located near to the N terminus of the solenoid and the other between the C-terminal juxtamembrane sequences. Moreover, Toll undergoes a ligand-induced conformational change, becoming more tightly curved than in the apo form. The unexpected 2:2 complex was confirmed by mass spectrometry under native conditions. These results suggest that activation of Toll is an allosteric mechanism induced by an end-on binding mode of its ligand Spz.


Subject(s)
Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Toll-Like Receptors/metabolism , Animals , Cell Line , Cryoelectron Microscopy , Dimerization , Drosophila Proteins/ultrastructure , Drosophila melanogaster/metabolism , Ligands , Protein Binding , Spodoptera
11.
J Cell Biol ; 173(6): 867-77, 2006 Jun 19.
Article in English | MEDLINE | ID: mdl-16785321

ABSTRACT

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p-centrin complexes containing several repeats show Sfi1p as an alpha helix with centrins wrapped around each repeat and similar centrin-centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p-centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous alpha helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p-centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.


Subject(s)
Calcium-Binding Proteins/chemistry , Cell Cycle Proteins/chemistry , Microtubule Proteins/chemistry , Repressor Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Spindle Apparatus/physiology , Binding Sites , Calcium-Binding Proteins/metabolism , Calcium-Binding Proteins/physiology , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/physiology , Crystallography, X-Ray , Mass Spectrometry , Microtubule Proteins/metabolism , Microtubule Proteins/physiology , Models, Molecular , Protein Binding , Protein Structure, Tertiary , Repetitive Sequences, Amino Acid , Repressor Proteins/metabolism , Repressor Proteins/physiology , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology , Sequence Analysis, Protein , Spindle Apparatus/ultrastructure
12.
Science ; 310(5754): 1658-61, 2005 Dec 09.
Article in English | MEDLINE | ID: mdl-16293722

ABSTRACT

We have examined the architecture of a protein complex in the absence of bulk water. By determining collision cross sections of assemblies of the trp RNA binding protein, TRAP, we established that the 11-membered ring topology of the complex can be maintained within a mass spectrometer. We also found that the binding of tryptophan enhances the stability of the ring structure and that addition of a specific RNA molecule increases the size of the complex and prevents structural collapse. These results provide definitive evidence that protein quaternary structure can be maintained in the absence of bulk water and highlight the potential of ion mobility separation for defining shapes of heterogeneous macromolecular assemblies.


Subject(s)
Bacterial Proteins/chemistry , Protein Structure, Quaternary , RNA-Binding Proteins/chemistry , Transcription Factors/chemistry , Water , 5' Untranslated Regions/metabolism , Apoproteins/chemistry , Apoproteins/metabolism , Bacillus subtilis , Bacterial Proteins/metabolism , Chemical Phenomena , Chemistry, Physical , Ions/chemistry , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA-Binding Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Thermodynamics , Transcription Factors/metabolism , Tryptophan/metabolism
13.
J Mol Biol ; 340(2): 373-83, 2004 Jul 02.
Article in English | MEDLINE | ID: mdl-15201058

ABSTRACT

Recent hydrogen-deuterium exchange experiments have highlighted tightening and loosening of protein structures upon ligand binding, with changes in bonding (DeltaH) and order (DeltaS) which contribute to the overall thermodynamics of ligand binding. Tightening and loosening show that ligand binding respectively stabilises or destabilises the internal structure of the protein, i.e. it shows positive or negative cooperativity between ligand binding and the receptor structure. In the case of membrane-bound receptors, such as G protein-coupled receptors (GPCRs) and ligand gated ion channel receptors (LGICRs), most binding studies have focussed on association/dissociation constants. Where these have been broken down into enthalpic and entropic contributions, the phenomenon of "thermodynamic discrimination" between antagonists and agonists has often been noted; e.g. for a receptor where agonist binding is predominantly enthalpy driven, antagonist binding is predominantly entropy driven and vice versa. These data have not previously been considered in terms of the tightening, or loosening, of receptor structures that respectively occurs upon positively, or negatively, cooperative binding of ligand. Nor have they been considered in light of the homo- and hetero-oligomerisation of GPCRs and the possibility of ligand-induced changes in oligomerisation. Here, we argue that analysis of the DeltaH and DeltaS of ligand binding may give useful information on ligand-induced changes in membrane-bound receptor oligomers, relevant to the differing effects of agonists and antagonists.


Subject(s)
Biopolymers/chemistry , Biopolymers/metabolism , Ligands , Protein Binding , Receptors, Cell Surface/agonists , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/metabolism
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