| 1. |
- Al-Amin, Rasel A., Researcher, 1983-, et al.
(författare)
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Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:22, s. e129-e129
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Tidskriftsartikel (refereegranskat)abstract
- Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.
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| 2. |
- Al-Amin, Rasel A., 1983-, et al.
(författare)
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Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in Situ
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins. In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins.
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| 3. |
- Blokzijl, Andries, et al.
(författare)
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Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundThe diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder.ObjectivesTo investigate SOX10 as a potential biomarker for melanoma and vitiligo.MethodsIn this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes.ResultsThe specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically.ConclusionsWe show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.
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| 4. |
- Blokzijl, Andries
(författare)
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Ligand-receptor interactions and signaling cross-talk in the TGF-b superfamily
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The transforming growth factor-beta (TGF-beta) superfamily of secreted polypeptide growth factors forms part of an evolutionary conserved signaling pathway, present in diverse organisms from flies to humans, used in intercellular communication. The TGF-beta family comprises more than 40 members in vertebrates, Caenorhabditis elegans and Drosophila melanogaster. Based on sequence similarities, this family can be divided into three main subfamilies, the TGF-betas, the activins, and the bone morphogenetic proteins (BMPs). Members of this family regulate a wide number of events during embryonic development and in adult life, including proliferation, apoptosis, differentiation and cell fate. The members of the TGF-beta family transmit signals through a receptor complex on the cell surface, containing two types (type I and II) of transmembrane receptor serine/threonine kinases (RSTKs). The receptors transmit the signal to a class of intracellular signaling intermediates, the Smad proteins, which then translocate to the nucleus where they interact with DNA and other transcription factors to regulate gene transcription. The aim of this thesis is to identify components involved in TGF-beta superfamily signal transduction, with special focus on molecules involved in signaling through the receptor activin receptor-like kinase 7 (ALK7). ALK7 is a type I receptor with a specific expression pattern in the nervous system, pancreas and brown fat. At the time the present work commenced, the cognate ligand, type II receptor, and downstream signaling pathways of ALK7 were unknown. Smad2 and Smad3 were initially established as intracellular mediators of ALK7. We then identified Xenopus Nodal-related 1 (Xnr1) and mouse Nodal proteins as ligands for ALK7, in collaboration with the type II receptor, ActRIlB, and the co-receptor, Cripto. Nodal proteins have instructive roles in mesendoderm formation and left-right patterning during vertebrate development, for which no receptor or downstream signal transduction mechanism were known. In search for novel interactors of Smad proteins, we discovered direct protein-protein interaction events between Smads and GATA-3, the Notch intracellular domain (NICD), and Phox2. GATA-3 is a zinc-finger transcription factor with a specific expression pattern in different neuronal subtypes and in the immune system, where it is an important regulator of T helper cell development. In particular, we have shown that TGF-beta upregulates interleukin-10 expression, in T helper cells in a GATA-3 dependent manner. The conserved Notch signal transduction pathway controls cell fate decisions in many different cell types, such as for example neural stem cells. Synergistic interactions between NICD and Smad3 were found to regulate the expression of Hes-1, a transcription factor known to be a direct target of the Notch pathway. By blocking Notch function using a dominant negative form of CSL, a DNAbinding co-factor of Notch, we could inhibit the ability of TGF-beta to upregulate Hes-1, demonstrating a direct link between the two pathways. The Phox2 proteins are homeodomain transcription factors with specific expression pattern in noradrenergic neurons, where they function as key regulators of the dopamine beta hydroxylase (DBH) gene. Both TGF-beta and BMP signaling has been implicated in DBH expression and we could show that this can in part be mediated through a direct interaction between Smads and Phox2 proteins. In conclusion this thesis work has characterized a signal transduction pathway from receptor-ligand interactions to transcriptional regulation, and the cross-talk with other signaling pathways.
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| 5. |
- Blokzijl, Andries, et al.
(författare)
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Profiling protein expression and interactions : proximity ligation as a tool for personalized medicine
- 2010
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Ingår i: Journal of Internal Medicine. - : Wiley. - 0954-6820 .- 1365-2796. ; 268:3, s. 232-245
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Forskningsöversikt (refereegranskat)abstract
- Blokzijl A, Friedman M, Ponten F, Landegren U (From the Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden). Profiling protein expression and interactions: proximity ligation as a tool for personalized medicine. (Review) J Intern Med 2010; 268: 232-245. The ability to detect very low levels of expressed proteins has enormous potential for early diagnostics and intervention at curable stages of disease. An extended range of targets such as interacting or post-translationally modified proteins can further improve the potential for diagnostics and patient stratification, and for monitoring response to treatment. These are critical building blocks for personalized treatment strategies to manage disease. The past few decades have seen a remarkably improved understanding of the molecular basis of disease in general, and of tumour formation and progression in particular. This accumulated knowledge creates opportunities to develop drugs that specifically target molecules or molecular complexes critical for survival and expansion of tumour cells. However, tumours are highly variable between patients, necessitating the development of diagnostic tools to individualize treatment through parallel analysis of sets of biomarkers. The proximity ligation assay (PLA) can address many of the requirements for advanced molecular analysis. The method builds on the principle that recognition of target proteins by two, three or more antibodies can bring in proximity DNA strands attached to the antibodies. The DNA strands can then participate in ligation reactions, giving rise to molecules that are amplified for highly sensitive detection. PLA is particularly well suited for sensitive, specific and multiplexed analysis of protein expression, post-translational modifications and protein-protein interactions. The analysis of this extended range of biomarkers will prove critical for the development and implementation of personalized medicine.
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| 6. |
- Blokzijl, Andries, et al.
(författare)
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Protein biomarker validation via proximity ligation assays
- 2014
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1844:5, s. 933-939
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Forskningsöversikt (refereegranskat)abstract
- The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PIA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. (C) 2013 Elsevier B.V. All rights reserved.
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| 7. |
- Blokzijl, Andries, et al.
(författare)
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Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens
- 2016
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 15:6, s. 1848-1856
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Tidskriftsartikel (refereegranskat)abstract
- The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA. Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.
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| 8. |
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| 9. |
- Gu, Gucci Jijuan, et al.
(författare)
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Role of Individual MARK Isoforms in Phosphorylation of Tau at Ser262 in Alzheimer's Disease
- 2013
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Ingår i: Neuromolecular medicine. - : Springer. - 1535-1084 .- 1559-1174. ; 15:3, s. 458-469
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Tidskriftsartikel (refereegranskat)abstract
- The microtubule-affinity regulating kinase (MARK) family consists of four highly conserved members that have been implicated in phosphorylation of tau protein, causing formation of neurofibrillary tangles in Alzheimer’s disease (AD). Understanding of roles by individual MARK isoform in phosphorylating tau has been limited due to lack of antibodies selective for each MARK isoform. In this study, we first applied the proximity ligation assay on cells to select antibodies specific for each MARK isoform. In cells, a CagA peptide specifically and significantly inhibited tau phosphorylation at Ser262 mediated by MARK4 but not other MARK isoforms. We then used these antibodies to study expression levels of MARK isoforms and interactions between tau and individual MARK isoforms in postmortem human brains. We found a strong and significant elevation of MARK4 expression and MARK4–tau interactions in AD brains, correlating with the Braak stages of the disease. These results suggest the MARK4–tau interactions are of functional importance in the progression of AD and the results also identify MARK4 as a promising target for AD therapy.
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| 10. |
- Shaw, Alan, et al.
(författare)
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Spatial control of membrane receptor function using ligand nanocalipers
- 2014
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Ingår i: Nature Methods. - : Springer Nature. - 1548-7091 .- 1548-7105. ; 11:8, s. 841-846
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Tidskriftsartikel (refereegranskat)abstract
- The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor mediated signaling.
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