scholarly journals A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2

2021 ◽  
Vol 7 (15) ◽  
pp. eabg1950
Author(s):  
Nicolas Bery ◽  
Carole J.R. Bataille ◽  
Angela Russell ◽  
Angela Hayes ◽  
Florence Raynaud ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
Binding Sites ◽  
Screening Method ◽  
Chromosomal Translocation ◽  
Resonance Method ◽  
Protein Protein Interactions ◽  
Intracellular Antibody ◽  
A Cell

Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody–guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.

scholarly journals Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
James O’Connell ◽  
John Porter ◽  
Boris Kroeplien ◽  
Tim Norman ◽  
Stephen Rapecki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Protein Interactions ◽  
General Mechanism ◽  
Protein Protein Interactions ◽  
Biologic Drugs ◽  
Small Molecule Drugs ◽  
Necrosis Factor

AbstractTumour 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.

Aryloxy Triester Phosphoramidates as Phosphoserine Biocleavable Masking Motifs

2019 ◽  
Author(s):  
Ageo Miccoli ◽  
Binar A. Dhiani ◽  
Peter J. Thornton ◽  
Olivia A. Lambourne ◽  
Edward James ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
In Silico ◽  
Parent Compound ◽  
Cellular Protein ◽  
Carboxypeptidase Y ◽  
Protein Protein Interactions ◽  
Enzymatic Assays ◽  
Specific Targeting

Many cellular protein-protein interactions (PPIs) are mediated by phosphoserine. The specific targeting of these PPIs by phosphoserine-containing small molecules has been scarce due to the dephosphorylation of phosphoserine and its charged nature at physiological pH, which hinders its uptake into cells. To address these issues, we herein report the masking of the phosphate group of phosphoserine with biocleavable aryloxy triester phosphoramidate groups. A combination of <i>in vitro</i> enzymatic assays and <i>in silico</i> studies, using carboxypeptidase Y and Hint-1 respectively, showed that the phosphate masking groups are metabolized to release phosphoserine. To probe the applicability of this phosphoserine masking approach, it was applied to a phosphoserine-containing inhibitor of 14-3-3 dimerization, and this generated molecules with improved pharmacological activity in cells compared to their unmasked phosphoserine-containing parent compound. Collectively, the data showcases the masking of phosphoserine with biocleavable aryloxy triester phosphoramidate masking groups as an efficient intracellular delivery system for phosphoserine-containing molecules.

scholarly journals A cell-free approach to accelerate the study of protein–protein interactions in vitro

2013 ◽  
Vol 3 (5) ◽  
pp. 20130018 ◽  
Author(s):  
E. Sierecki ◽  
N. Giles ◽  
M. Polinkovsky ◽  
M. Moustaqil ◽  
K. Alexandrov ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Single Molecule ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Interaction Networks ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Single Molecule Fluorescence Spectroscopy ◽  
A Cell

Protein–protein interactions are highly desirable targets in drug discovery, yet only a fraction of drugs act as binding inhibitors. Here, we review the different technologies used to find and validate protein–protein interactions. We then discuss how the novel combination of cell-free protein expression, AlphaScreen and single-molecule fluorescence spectroscopy can be used to rapidly map protein interaction networks, determine the architecture of protein complexes, and find new targets for drug discovery.

Aryloxy Triester Phosphoramidates as Phosphoserine Biocleavable Masking Motifs

2019 ◽  
Author(s):  
Ageo Miccoli ◽  
Binar A. Dhiani ◽  
Peter J. Thornton ◽  
Olivia A. Lambourne ◽  
Edward James ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
In Silico ◽  
Parent Compound ◽  
Cellular Protein ◽  
Carboxypeptidase Y ◽  
Protein Protein Interactions ◽  
Enzymatic Assays ◽  
Specific Targeting

Many cellular protein-protein interactions (PPIs) are mediated by phosphoserine. The specific targeting of these PPIs by phosphoserine-containing small molecules has been scarce due to the dephosphorylation of phosphoserine and its charged nature at physiological pH, which hinders its uptake into cells. To address these issues, we herein report the masking of the phosphate group of phosphoserine with biocleavable aryloxy triester phosphoramidate groups. A combination of <i>in vitro</i> enzymatic assays and <i>in silico</i> studies, using carboxypeptidase Y and Hint-1 respectively, showed that the phosphate masking groups are metabolized to release phosphoserine. To probe the applicability of this phosphoserine masking approach, it was applied to a phosphoserine-containing inhibitor of 14-3-3 dimerization, and this generated molecules with improved pharmacological activity in cells compared to their unmasked phosphoserine-containing parent compound. Collectively, the data showcases the masking of phosphoserine with biocleavable aryloxy triester phosphoramidate masking groups as an efficient intracellular delivery system for phosphoserine-containing molecules.

scholarly journals Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

1986 ◽  
Vol 6 (12) ◽  
pp. 4168-4178 ◽  
Author(s):  
C L Peterson ◽  
K Orth ◽  
K L Calame
Keyword(s):  
Heavy Chain ◽  
Protein Interactions ◽  
Binding Sites ◽  
Immunoglobulin Heavy Chain ◽  
Lymphoid Cells ◽  
Protein Protein Interactions ◽  
Protein Activity ◽  
Enhancer Element ◽  
Enhancer Sequences

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.

scholarly journals Norstictic acid is a selective allosteric transcriptional regulator

2021 ◽  
Author(s):  
Julie M Garlick ◽  
Steven M Sturlis ◽  
Paul A Bruno ◽  
Joel A Yates ◽  
Amanda Peiffer ◽  
...  
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Triple Negative ◽  
Transcriptional Coactivator ◽  
Protein Protein Interactions ◽  
Ppi Networks ◽  
Binding Surface ◽  
Inhibitor Discovery

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (~900 square angstroms) and have little topology, and thus do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprised of a highly dynamic loop flanking one canonical binding surface and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.

scholarly journals Synthesis of Novel Suramin Analogs With Anti-Proliferative Activity via FGF1 and FGFRD2 Blockade

2022 ◽  
Vol 9 ◽  
Author(s):  
Nuzhat Parveen ◽  
Yan-Liang Lin ◽  
Ruey-Hwang Chou ◽  
Chung-Ming Sun ◽  
Chin Yu
Keyword(s):  
Cell Proliferation ◽  
Protein Interactions ◽  
Binding Sites ◽  
Proliferative Activity ◽  
Three Dimensional ◽  
Cancer Cell Line ◽  
Protein Protein Interactions ◽  
Docking Simulations ◽  
Three Dimensional Models

A promising approach in cancer therapy is the inhibition of cell proliferation using small molecules. In this study, we report the synthesis of suramin derivatives and their applications. We used NMR spectroscopy and docking simulations to confirm binding sites and three-dimensional models of the ligand-protein complex. The WST-1 assay was used to assess cell viability and cell proliferation in vitro to evaluate the inhibition of protein–protein interactions and to investigate the anti-proliferative activities in a breast cancer cell line. All the suramin derivatives showed anti-proliferative activity by blocking FGF1 binding to its receptor FGFRD2. The dissociation constant was measured by fluorescence spectroscopy. The suramin compound derivatives synthesized herein show potential as novel therapeutic agents for their anti-proliferative activity via the inhibition of protein–protein interactions. The cytotoxicity of these suramin derivatives was lower than that of the parent suramin compound, which may be considered a significant advancement in this field. Thus, these novel suramin derivatives may be considered superior anti-metastasis molecules than those of suramin.

scholarly journals Emerging strategies for the identification of protein–metabolite interactions

2019 ◽  
Vol 70 (18) ◽  
pp. 4605-4618 ◽  
Author(s):  
Marcin Luzarowski ◽  
Aleksandra Skirycz
Keyword(s):  
Ligand Binding ◽  
Small Molecules ◽  
Protein Interactions ◽  
Technological Progress ◽  
Molecular Complexes ◽  
Biological Molecules ◽  
Protein Protein Interactions ◽  
Binding Domains ◽  
Protein Receptors ◽  
A Cell

AbstractInteractions between biological molecules enable life. The significance of a cell-wide understanding of molecular complexes is thus obvious. In comparison to protein–protein interactions, protein–metabolite interactions remain under-studied. However, this has been gradually changing due to technological progress. Here, we focus on the interactions between ligands and receptors, the triggers of signalling events. While the number of small molecules with proven or proposed signalling roles is rapidly growing, most of their protein receptors remain unknown. Conversely, there are numerous signalling proteins with predicted ligand-binding domains for which the identities of the metabolite counterparts remain elusive. Here, we discuss the current biochemical strategies for identifying protein–metabolite interactions and how they can be used to characterize known metabolite regulators and identify novel ones.

Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA

1986 ◽  
Vol 6 (12) ◽  
pp. 4168-4178
Author(s):  
C L Peterson ◽  
K Orth ◽  
K L Calame
Keyword(s):  
Heavy Chain ◽  
Protein Interactions ◽  
Binding Sites ◽  
Immunoglobulin Heavy Chain ◽  
Lymphoid Cells ◽  
Protein Protein Interactions ◽  
Protein Activity ◽  
Enhancer Element ◽  
Enhancer Sequences

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.

scholarly journals Structural Modeling of Cell Wall Peptidase CwpFM (EntFM) Reveals Distinct Intrinsically Disordered Extensions Specific to Pathogenic Bacillus cereus Strains

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 593
Author(s):  
Seav-Ly Tran ◽  
Delphine Cormontagne ◽  
Jasmina Vidic ◽  
Gwenaëlle André-Leroux ◽  
Nalini Ramarao
Keyword(s):  
Cell Wall ◽  
Protein Interactions ◽  
Health Concern ◽  
Protein Protein Interactions ◽  
Src Homology 3 ◽  
Intrinsically Disordered ◽  
Food Borne ◽  
A Cell ◽  
Src Homology

The emergence of B. cereus as an opportunistic food-borne pathogen has intensified the need to distinguish strains of public health concern. The heterogeneity of the diseases associated with B. cereus infections emphasizes the versatility of these bacteria strains to colonize their host. Nevertheless, the molecular basis of these differences remains unclear. Several toxins are involved in virulence, particularly in gastrointestinal disorders, but there are currently no biological markers able to differentiate pathogenic from harmless strains. We have previously shown that CwpFM is a cell wall peptidase involved in B. cereus virulence. Here, we report a sequence/structure/function characterization of 39 CwpFM sequences, chosen from a collection of B. cereus with diverse virulence phenotypes, from harmless to highly pathogenic strains. CwpFM is homology-modeled in silico as an exported papain-like endopeptidase, with an N-terminal end composed of three successive bacterial Src Homology 3 domains (SH3b1–3) likely to control protein–protein interactions in signaling pathways, and a C-terminal end that contains a catalytic NLPC_P60 domain primed to form a competent active site. We confirmed in vitro that CwpFM is an endopeptidase with a moderate peptidoglycan hydrolase activity. Remarkably, CwpFMs from pathogenic strains harbor a specific stretch of twenty residues intrinsically disordered, inserted between the SH3b3 and the catalytic NLPC_P60 domain. This strongly suggests this linker as a marker of differentiation between B. cereus strains. We believe that our findings improve our understanding of the pathogenicity of B. cereus while advancing both clinical diagnosis and food safety.

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