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MicroScale Thermophoresis

Andrew M Prantner, Catherine Yin, Kalika Kamat, Khushboo Sharma, Andrew C Lowenthal, Peter Bawden Madrid, Nathalie Scholler
Mesothelin is an epithelial marker highly expressed at the cell surface of cancer cells from diverse origins, including ovarian and pancreatic adenocarcinomas and mesotheliomas. Previously, we identified and characterized an anti-mesothelin nanobody (NbG3a) for in vitro diagnostic applications. The main goal of this research was to establish the potential of NbG3a as a molecular imaging agent. Site-specific biotinylated NbG3a (bNbG3a) was bound to streptavidin-conjugated reagents for in vitro and in vivo assays...
February 20, 2018: Molecular Pharmaceutics
Julie M Rainard, George C Pandarakalam, Stuart P McElroy
High-throughput screening (HTS) is a proven method for discovering new lead matter for drug discovery and chemical biology. To maximize the likelihood of identifying genuine binders to a molecular target, and avoid wasting resources following up compounds with unproductive/nonspecific mechanisms of action, it is important to employ a range of assays during an HTS campaign that build confidence of target engagement for hit compounds. Biophysical methods that measure direct target/compound engagement have established themselves as key techniques in generating this confidence, and they are now integral to the latter stages of HTS triage at the European Lead Factory (ELF)...
March 2018: SLAS Discovery
Yiwen Zhang, Zhimei Li, Qiuxia Min, Abulizi Palida, Yiyuan Zhang, Ruotian Tang, Lixia Chen, Hua Li
8-Chrysoeriol, a bioactive flavanoid, was firstly identified to bind directly to BCL-2 as BH3 mimetics by structure-based virtual ligand screening. And 3D docking model revealed the molecular basis of 8-Chrysoeriol targeting to BCL-2. The interaction between 8-Chrysoeriol and BCL-2 was further confirmed using Microscale Thermophoresis (MST) technique. Meanwhile, high expression level of antiapoptotic protein BCL-2 was detected in SW1990 pancreatic cancer cells and 8-Chrysoeriol showed obvious proapoptosis effect against SW1990 in vitro...
February 2, 2018: Bioorganic Chemistry
Mufarreh Asmari, Ratih Ratih, Hassan A Alhazmi, Sami El Deeb
The study of biomolecular interactions is crucial to get more insight into the biological system. The interactions of protein-protein, protein-nucleic acids, protein-sugars, nucleic acid-nucleic acids and protein-small molecules are supporting therapeutics and technological developments. Recently, the development in a large number of analytical techniques for characterizing biomolecular interactions reflect the promising research investments in this field. In this review, microscale thermophoresis technology (MST) is presented as an analytical technique for characterizing biomolecular interactions...
February 10, 2018: Methods: a Companion to Methods in Enzymology
Marta Narczyk, Branimir Bertoša, Lucija Papa, Vedran Vuković, Ivana Leščić Ašler, Beata Wielgus-Kutrowska, Agnieszka Bzowska, Marija Luić, Zoran Štefanić
Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (Pi ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies...
February 12, 2018: FEBS Journal
Chuannan Long, Mengmeng Liu, Xia Chen, Xiaofang Wang, Mingqiang Ai, Jingjing Cui, Bin Zeng
The present study verified whether acyl-coenzyme A (acyl-CoA)-binding protein (ACBP) affected the production of Monascus pigments (MPs) in Monascus ruber CICC41233 (MrACBP). Phylogenetic analysis revealed that the cloned Mracbp gene, which encoded the MrACBP protein, exhibited the closest match (99% confidence level) to the gene from Penicilliopsis zonata . The MrACBP and maltose-binding protein (MBP) were simultaneously expressed in Escherichia coli Rosetta DE3 in the form of a fusion protein. The microscale thermophoresis binding assay revealed that the purified MBP-MrACBP exhibited a higher affinity for myristoyl-CoA (Kd = 88...
February 2018: 3 Biotech
Michael Zepp, Marineta Kovacheva, Munkhtsetseg Altankhuyag, Gabriela Westphal, Irina Berger, Katharina S Gather, Heidegard Hilbig, Jochen Neuhaus, Gertrud M Hänsch, Franz P Armbruster, Martin R Berger
Changes in glycosylation are salient features of cancer cells. Here, we report on the diagnostic and therapeutic properties of IDK1, an antibody against tumour associated, hypoglycosylated bone sialoprotein (hypo-BSP). The affinity of the rat monoclonal antibody IDK1 for hypo-BSP, as determined by microscale thermophoresis, was three orders of magnitude higher than for mature BSP, whereas the mouse monoclonal antibody used had similar affinity for both BSP forms. IDK1 showed no activity against the proliferation or migration of normal or cancer cells growing in vitro...
January 2018: Journal of Pathology. Clinical Research
Anmol S Adhav, Surabhi R Kokane, Rakesh S Joshi
Trehalase catalyzes hydrolysis of trehalose and plays a crucial role in insect metabolism. In the present study, phylogenetic analysis and multiple sequence alignment suggested that H. armigera trehalase-1 (HaTre-1) is closely related to other soluble trehalases with conserved signature features and functional sites. We have expressed and purified recombinant HaTre-1 having Vmax ~0.16 mM/min and Km ~1.34 mM. Inhibition kinetics and Microscale thermophoresis illustrated competitive inhibition of HaTre-1 by Validamycin A having Ki ~3 nM and KD ~542 nM, respectively...
February 2, 2018: International Journal of Biological Macromolecules
Xiaoyu Shi, Yue Sun, Ping Wang, Lingling Gu, Lu Wang, Huan Yang, Qun Wei, Zhimei Li, Jing Luo
Calcineurin (CN) is a protein phosphatase and widely distributed in eukaryotes, with an extremely high level of expression in mammalian brain. Alpha-synuclein (α-syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. In our present study, we explored the interactions between CN and α-syn in vitro. Based on the data from microscale thermophoresis, GST pull-down assays, and co-immunoprecipitation, we found that CN binds α-syn. Furthermore, this interaction is mediated by calcium/calmodulin (Ca2+/CaM) signaling...
January 30, 2018: Biochemical and Biophysical Research Communications
Canrong Wu, Mengzhu Zheng, Suyu Gao, Shanshan Luan, Li Cheng, Liqing Wang, Jiachen Li, Lixia Chen, Hua Li
Kidney-type glutaminase (KGA), a mitochondrial enzyme converting glutamine to glutamate for energy supply, was over-expressed in many cancers and had been regarded as a promising therapeutic target in recent years. Structure-based virtual ligand screening predicted physapubescin K, a new withanolide from Physalis pubescens, to be potential KGA inhibitor. Enzyme activity inhibition assays and microscale thermophoresis experiments had demonstrated the efficiency and specificity of physapubescin K targeting KGA...
December 26, 2017: Oncotarget
Hyun Wook Seo, Joon Pyung Seo, Yoon Jun Kim, Guhung Jung
Hepatitis B virus (HBV) infection is a major risk factor for chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) worldwide. While multiple hepatitis B drugs have been developed, development of drug resistance during treatment or weak efficacies observed in some cases have limited their application. Therefore, there is an urgent need to develop better pharmacological agents for HBV-infected individuals. Here, we identified cetylpyridinium chloride (CPC) as a novel inhibitor of HBV. Using computational analysis of CPC-nucleocapsid proteins, microscale thermophoresis analysis of CPC binding to viral nucleocapsids, and in vitro nucleocapsid formation assays, we found that CPC interacts with dimeric viral nucleocapsid protein (known as core protein or HBcAg)...
January 15, 2018: Biochemical and Biophysical Research Communications
Vladimir F Lazarev, Dmitry V Sverchinsky, Elena R Mikhaylova, Pavel I Semenyuk, Elena Y Komarova, Sergey A Niskanen, Alina D Nikotina, Anton V Burakov, Viktor G Kartsev, Irina V Guzhova, Boris A Margulis
Hsp70 chaperone controls proteostasis and anti-stress responses in rapidly renewing cancer cells, making it an important target for therapeutic compounds. To date several Hsp70 inhibitors are presented with remarkable anticancer activity, however their clinical application is limited by the high toxicity towards normal cells. This study aimed to develop assays to search for the substances that reduce the chaperone activity of Hsp70 and diminish its protective function in cancer cells. On our mind the resulting compounds alone should be safe and function in combination with drugs widely employed in oncology...
January 18, 2018: Cell Death & Disease
Yanli Xie, Xiaolu Zhu, Yuan Li, Cheng Wang
The Fe(III) chelating activity of anthocyanin extracted from black soybean coats was investigated at pH 3.0, 5.0, 6.5, 7.0 and 7.4 with fluorescence spectroscopy and microscale thermophoresis (MST). Cyanidin-3-glucoside (C3G) was determined to be 98% of the total anthocyanin by HPLC. The binding affinity (Ka) exhibited significant pH-dependent behavior; Ka was 9.7167, 1.0837, 1.4284, 5.4550 and 3.0269×104 M-1 at pH 3.0, 5.0, 6.5, 7.0 and 7.4, respectively (p<0.05). The MST data showed that ΔG<0 and ΔH<0, demonstrating that chelation is spontaneous and exothermic...
January 15, 2018: Journal of Agricultural and Food Chemistry
Michelle H Moon, Thomas Hilimire, Allix M Sanders, John S Schneekloth
In the past twenty years, there has been a dramatic increase in the study of RNA. RNA has gone from being known as an intermediate in the central dogma of molecular biology to a molecule with a large diversity of structure and function that is involved in all aspects of biology. As new functions are rapidly discovered, it has become clear that there is a need for RNA-targeting small molecule probes to investigate RNA biology and clarify the potential for therapeutics based on RNA/small molecule interactions...
January 12, 2018: Biochemistry
Torun Ekblad, Patricia Verheugd, Anders E Lindgren, Tomas Nyman, Mikael Elofsson, Herwig Schüler
Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay...
January 1, 2018: SLAS Discovery
Fengyan Bao, Kaiyin Yang, Canrong Wu, Suyu Gao, Penghe Wang, Lixia Chen, Hua Li
Hexokinase 2 (HK2), a rate-limiting enzyme in the first step of glycolysis pathway, expresses at high level in cancer cells compared with normal cells. HK2 provides a new target for cancer therapy due to its pivotal role in tumor tumourigenic and metastatic process. The structure-based virtual ligand screening in a small in-house database of natural products predicted that a new steroid, (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (2) from Ganoderma sinense has high binding affinity to HK2 with significant calculated binding free energy...
January 3, 2018: Fitoterapia
Romain Magnez, Bryan Thiroux, Solenne Taront, Zacharie Segaoula, Bruno Quesnel, Xavier Thuru
The characterization of protein interactions has become essential in many fields of life science, especially drug discovery. Microscale thermophoresis (MST) is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. In addition, one of the major advantages of this technique is that no tedious purification step is necessary to access the protein of interest. Here, we describe a protocol using MST to determine the binding affinity of the PD-1/PD-L1 couple, which is involved in tumour escape processes, without purification of the target protein from cell lysates...
December 15, 2017: Scientific Reports
Damien Morger, Franziska Zosel, Martin Bühlmann, Sara Züger, Maximilian Mittelviefhaus, Benjamin Schuler, Jeremy Luban, Markus G Grütter
Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remains largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro This is thought to be due to differences in binding to the capsid lattice...
December 13, 2017: Journal of Virology
Maria-Jose Garcia-Bonete, Maja Jensen, Christian V Recktenwald, Sandra Rocha, Volker Stadler, Maria Bokarewa, Gergely Katona
A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners...
December 1, 2017: Scientific Reports
Lorenz M R Keil, Friederike M Möller, Michael Kieß, Patrick W Kudella, Christof B Mast
Proton gradients are essential for biological systems. They not only drive the synthesis of ATP, but initiate molecule degradation and recycling inside lysosomes. However, the high mobility and permeability of protons through membranes make pH gradients very hard to sustain in vitro. Here we report that heat flow across a water-filled chamber forms and sustains stable pH gradients. Charged molecules accumulate by convection and thermophoresis better than uncharged species. In a dissociation reaction, this imbalances the reaction equilibrium and creates a difference in pH...
December 1, 2017: Nature Communications
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