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

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.

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Structure-aided prediction of mammalian transcription factor complexes in conserved non-coding elements

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0029 ◽

2013 ◽

Vol 368 (1632) ◽

pp. 20130029 ◽

Cited By ~ 24

Author(s):

Harendra Guturu ◽

Andrew C. Doxey ◽

Aaron M. Wenger ◽

Gill Bejerano

Keyword(s):

Transcription Factor ◽

Binding Site ◽

Protein Interactions ◽

Binding Sites ◽

Three Dimensional ◽

Structural Data ◽

Regulatory Elements ◽

Computational Method ◽

Web Resource ◽

Three Dimensional Models

Mapping the DNA-binding preferences of transcription factor (TF) complexes is critical for deciphering the functions of cis -regulatory elements. Here, we developed a computational method that compares co-occurring motif spacings in conserved versus unconserved regions of the human genome to detect evolutionarily constrained binding sites of rigid TF complexes. Structural data were used to estimate TF complex physical plausibility, explore overlapping motif arrangements seldom tackled by non-structure-aware methods, and generate and analyse three-dimensional models of the predicted complexes bound to DNA. Using this approach, we predicted 422 physically realistic TF complex motifs at 18% false discovery rate, the majority of which (326, 77%) contain some sequence overlap between binding sites. The set of mostly novel complexes is enriched in known composite motifs, predictive of binding site configurations in TF–TF–DNA crystal structures, and supported by ChIP-seq datasets. Structural modelling revealed three cooperativity mechanisms: direct protein–protein interactions, potentially indirect interactions and ‘through-DNA’ interactions. Indeed, 38% of the predicted complexes were found to contain four or more bases in which TF pairs appear to synergize through overlapping binding to the same DNA base pairs in opposite grooves or strands. Our TF complex and associated binding site predictions are available as a web resource at http://bejerano.stanford.edu/complex .

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

Science Advances ◽

10.1126/sciadv.abg1950 ◽

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.

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Interactions of invadopodia scaffold protein TKS4 with intersectin family proteins

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v25.1152 ◽

2019 ◽

Vol 25 ◽

pp. 126-130

Author(s):

S. V. Kropyvko ◽

T. A. Gryaznova ◽

A. V. Rynditch

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Cancer Cell Line ◽

Adaptor Proteins ◽

Metastatic Potential ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Membrane Structures ◽

Active Forms Of Oxygen

Aim. TKS4 is one of the key proteins of invadopodies, specialized membrane structures that provide invasiveness and metastatic potential of malignant cells. This protein is a substrate for the tyrosine kinase SRC, which is involved in the formation of the membrane bends, affects cellular production of active forms of oxygen, interacts with membrane metallоproteinases causing degradation of the extracellular matrix, but its role in invasive structures has not yet been fully understood. Further study of molecular components of invadopodies and their specific interactions provides better understanding of mechanisms of cellular invasion. Methods. Protein-protein interactions were identified by in vitro precipitation of protein complexes using GST-fused proteins and co-immunoprecipitation. Results. Adapter proteins ITSN1 and ITSN2 are new partners of TKS4. Interactions between intersectins and TKS4 are mediated by the SH3A, SH3C and SH3E domains of ITSN1 or ITSN2. TKS4 phosphorylation on tyrosine residues does not affect interactions between TKS4 and intersectins. Conclusions. In this study, we have showed that TKS4 interacts with endocytic adaptor proteins of the intersectin family, ITSN1 and ITSN2. We have also demonstrated that TKS4 and ITSN1 can form a complex in invasive MDA-MB-231 breast cancer cell line. Keywords: TKS4, ITSN1, ITSN2, protein-protein interactions.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4168 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4168-4178 ◽

Cited By ~ 22

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.

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Norstictic acid is a selective allosteric transcriptional regulator

10.1101/2021.03.26.437253 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4168-4178.1986 ◽

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

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target

Biomolecules ◽

10.3390/biom11040510 ◽

2021 ◽

Vol 11 (4) ◽

pp. 510

Author(s):

Maho Yamamoto ◽

Rina Kondo ◽

Haruka Hozumi ◽

Seita Doi ◽

Miwako Denda ◽

...

Keyword(s):

Protein Interactions ◽

Biochemical Characterization ◽

S100 Protein ◽

High Mobility ◽

Dependent Manner ◽

Protein Signaling ◽

Protein Protein Interactions ◽

High Mobility Group Protein ◽

Pulldown Assay

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

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Ways into Understanding HIF Inhibition

Cancers ◽

10.3390/cancers13010159 ◽

2021 ◽

Vol 13 (1) ◽

pp. 159

Author(s):

Tina Schönberger ◽

Joachim Fandrey ◽

Katrin Prost-Fingerle

Keyword(s):

Protein Interactions ◽

Target Genes ◽

Protein Protein Interactions ◽

Low Oxygen ◽

Drug Candidates ◽

Open Questions ◽

Wide Range ◽

Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

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EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

British Journal of Cancer ◽

10.1038/s41416-020-01250-4 ◽

2021 ◽

Author(s):

Liqing Jia ◽

Xiaolu Ge ◽

Chao Du ◽

Linna Chen ◽

Yanhong Zhou ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Protein Interactions ◽

Elongation Factor ◽

Protein Translation ◽

Protein Protein Interactions ◽

Promising Target ◽

Tgfβ Receptor ◽

Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

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