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ITC drug discovery

Judith E Unterlass, Arnaud Baslé, Timothy J Blackburn, Julie Tucker, Céline Cano, Martin E M Noble, Nicola J Curtin
3-Phosphoglycerate dehydrogenase (PHGDH) has recently been identified as an attractive target in cancer therapy as it links upregulated glycolytic flux to increased biomass production in cancer cells. PHGDH catalyses the first step in the serine synthesis pathway and thus diverts glycolytic flux into serine synthesis. We have used siRNA-mediated suppression of PHGDH expression to show that PHGDH is a potential therapeutic target in PHGDH-amplified breast cancer. Knockdown caused reduced proliferation in the PHGDH-amplified cell line MDA-MB-468, whereas breast cancer cells with low PHGDH expression or with elevated PHGDH expression in the absence of genomic amplification were not affected...
August 22, 2016: Oncotarget
Franziska U Huschmann, Janina Linnik, Karine Sparta, Monika Ühlein, Xiaojie Wang, Alexander Metz, Johannes Schiebel, Andreas Heine, Gerhard Klebe, Manfred S Weiss, Uwe Mueller
Crystallographic screening of the binding of small organic compounds (termed fragments) to proteins is increasingly important for medicinal chemistry-oriented drug discovery. To enable such experiments in a widespread manner, an affordable 96-compound library has been assembled for fragment screening in both academia and industry. The library is selected from already existing protein-ligand structures and is characterized by a broad ligand diversity, including buffer ingredients, carbohydrates, nucleotides, amino acids, peptide-like fragments and various drug-like organic compounds...
May 2016: Acta Crystallographica. Section F, Structural Biology Communications
Luca Mazzei, Stefano Ciurli, Barbara Zambelli
Isothermal titration calorimetry (ITC) is a technique that measures the heat released or absorbed during a chemical reaction as an intrinsic probe to characterize any chemical process that involves heat changes spontaneously occurring during the reaction. The general features of this method to determine the kinetic and thermodynamic parameters of enzymatic reactions (kcat, KM, ΔH) are described and discussed here together with some detailed applications to specific cases. ITC does not require any modification or labeling of the system under analysis, can be performed in solution, and needs only small amounts of enzyme...
2016: Methods in Enzymology
Javier Alguacil, Jordi Robles, Clara Ràfols, Elisabeth Bosch
Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity...
April 2016: Journal of Molecular Recognition: JMR
Prakash B Palde, Ashima Bhaskar, Laura E Pedró Rosa, Franck Madoux, Peter Chase, Vinayak Gupta, Timothy Spicer, Louis Scampavia, Amit Singh, Kate S Carroll
Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters...
January 15, 2016: ACS Chemical Biology
Hannah Wapenaar, Petra E van der Wouden, Matthew R Groves, Dante Rotili, Antonello Mai, Frank J Dekker
Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate...
November 13, 2015: European Journal of Medicinal Chemistry
Shanshan Qin, Yiran Ren, Xu Fu, Jie Shen, Xin Chen, Quan Wang, Xin Bi, Wenjing Liu, Lixin Li, Guangxin Liang, Cheng Yang, Wenqing Shui
Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidate's efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K(d) determination through UF-LC/MS by comparison with classical ITC measurement...
July 30, 2015: Analytica Chimica Acta
Vijay Soni, Priyanka Suryadevara, Dharmarajan Sriram, Santhosh Kumar, Vinay Kumar Nandicoori, Perumal Yogeeswari
Persistent nature of Mycobacterium tuberculosis is one of the major factors which make the drug development process monotonous against this organism. The highly lipophilic cell wall, which constituting outer mycolic acid and inner peptidoglycan layers, acts as a barrier for the drugs to enter the bacteria. The rigidity of the cell wall is imparted by the peptidoglycan layer, which is covalently linked to mycolic acid by arabinogalactan. Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) serves as the starting material in the biosynthesis of this peptidoglycan layers...
July 2015: Journal of Molecular Modeling
Fredrik Edfeldt, Johan Evenäs, Matti Lepistö, Alison Ward, Jens Petersen, Lisa Wissler, Mattias Rohman, Ulf Sivars, Karin Svensson, Matthew Perry, Isabella Feierberg, Xiao-Hong Zhou, Thomas Hansson, Frank Narjes
Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified...
June 15, 2015: Bioorganic & Medicinal Chemistry Letters
Rafael J Rojas, Dale E Edmondson, Terri Almos, Roderick Scott, Mark E Massari
Monoamine oxidase B (MAO-B) plays a key role in the metabolism of dopamine, a neurotransmitter critical for the maintenance of cognitive function. Consequently, MAO-B is an important therapeutic target for disorders characterized by a decline in dopaminergic neurotransmission, including Parkinson's disease (PD). An emerging strategy in drug discovery is to utilize the biophysical approaches of thermal shift and isothermal titration calorimetry (ITC) to gain insight into binding modality and identify thermodynamically privileged chemical scaffolds...
February 15, 2015: Bioorganic & Medicinal Chemistry
Yosup Rew, Daqing Sun, Xuelei Yan, Hilary P Beck, Jude Canon, Ada Chen, Jason Duquette, John Eksterowicz, Brian M Fox, Jiasheng Fu, Ana Z Gonzalez, Jonathan Houze, Xin Huang, Min Jiang, Lixia Jin, Yihong Li, Zhihong Li, Yun Ling, Mei-Chu Lo, Alexander M Long, Lawrence R McGee, Joel McIntosh, Jonathan D Oliner, Tao Osgood, Anne Y Saiki, Paul Shaffer, Yu Chung Wang, Sarah Wortman, Peter Yakowec, Qiuping Ye, Dongyin Yu, Xiaoning Zhao, Jing Zhou, Julio C Medina, Steven H Olson
Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2...
December 26, 2014: Journal of Medicinal Chemistry
Vivek Kumar, Abhigyan Sengupta, Krishna Gavvala, Raj Kumar Koninti, Partha Hazra
The G-quadruplex (GQ-DNA), an alternative structure motif of DNA, has emerged as a novel and exciting target for anticancer drug discovery. GQ-DNA formed in the presence of monovalent cations (Na(+)/K(+)) by human telomeric DNA is a point of interest due to their direct relevance for cellular aging and abnormal cell growths. Small molecules that selectively target and stabilize G-quadruplex structures are considered to be potential therapeutic anticancer agents. Herein, we probe G-quadruplex and proflavine (a well-known DNA intercalator, hence acting as an anticarcinogen) association through steady state and time-resolved fluorescence spectroscopy to explore the effect of stabilization of GQ-DNA by this well-known DNA intercalator...
September 25, 2014: Journal of Physical Chemistry. B
Omid Tavassoly, Joe Kakish, Sergiy Nokhrin, Oleg Dmitriev, Jeremy S Lee
A major feature of Parkinson's disease is the formation of Lewy bodies in dopaminergic neurons which consist of misfolded α-synuclein. The binding of natural products to α-synuclein was evaluated by nanopore analysis and caffeine, curcumin, and nicotine all caused large conformational changes which may be related to their known neuroprotective effect in Parkinson's disease. The binding of the stereoisomers of nicotine were also studied by ITC, CD and NMR. It is proposed that (-)-nicotine causes the folding of α-synuclein into a loop with interaction between the N- and C-termini...
December 17, 2014: European Journal of Medicinal Chemistry
Luca Mazzei, Stefano Ciurli, Barbara Zambelli
Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (kcat, KM, ΔH) of enzymatic reactions, even though this application is still underexploited...
2014: Journal of Visualized Experiments: JoVE
Igor M Prokopczyk, Jean F R Ribeiro, Geraldo R Sartori, Renata Sesti-Costa, João S Silva, Renato F Freitas, Andrei Leitão, Carlos A Montanari
BACKGROUND: The enzyme gapdh, which acts in the glycolytic pathway, is seen as a potential target for pharmaceutical intervention of chagas disease. RESULTS: Herein, we report the discovery of new Trypanosoma cruzi GAPDH (TcGAPDH) inhibitors from target- and ligand-based virtual screening protocols using isothermal titration calorimetry (ITC) and molecular dynamics. Molecular dynamics simulations were used to gain insight on the binding poses of newly identified inhibitors acting at the TcGAPDH substrate (G3P) site...
January 2014: Future Medicinal Chemistry
Ellene H Mashalidis, Paweł Śledź, Steffen Lang, Chris Abell
This protocol describes the screening of a library of low-molecular-weight compounds (fragments) using a series of biophysical ligand-binding assays. Fragment-based drug discovery (FBDD) has emerged as a successful method to design high-affinity ligands for biomacromolecules of therapeutic interest. It involves detecting relatively weak interactions between the fragments and a target macromolecule using sensitive biophysical techniques. These weak binders provide a starting point for the development of inhibitors with submicromolar affinity...
November 2013: Nature Protocols
Juan Carlos Pizarro, Tanya Hills, Guillermo Senisterra, Amy K Wernimont, Claire Mackenzie, Neil R Norcross, Michael A J Ferguson, Paul G Wyatt, Ian H Gilbert, Raymond Hui
Human African trypanosomiasis is a neglected parasitic disease that is fatal if untreated. The current drugs available to eliminate the causative agent Trypanosoma brucei have multiple liabilities, including toxicity, increasing problems due to treatment failure and limited efficacy. There are two approaches to discover novel antimicrobial drugs--whole-cell screening and target-based discovery. In the latter case, there is a need to identify and validate novel drug targets in Trypanosoma parasites. The heat shock proteins (Hsp), while best known as cancer targets with a number of drug candidates in clinical development, are a family of emerging targets for infectious diseases...
2013: PLoS Neglected Tropical Diseases
Bo Wang, Liwei Li, Thomas D Hurley, Samy O Meroueh
End-point free energy calculations using MM-GBSA and MM-PBSA provide a detailed understanding of molecular recognition in protein-ligand interactions. The binding free energy can be used to rank-order protein-ligand structures in virtual screening for compound or target identification. Here, we carry out free energy calculations for a diverse set of 11 proteins bound to 14 small molecules using extensive explicit-solvent MD simulations. The structure of these complexes was previously solved by crystallography and their binding studied with isothermal titration calorimetry (ITC) data enabling direct comparison to the MM-GBSA and MM-PBSA calculations...
October 28, 2013: Journal of Chemical Information and Modeling
Veronika Škedelj, Andrej Perdih, Matjaž Brvar, Ana Kroflič, Vincent Dubbée, Victoria Savage, Alex J O'Neill, Tom Solmajer, Marija Bešter-Rogač, Didier Blanot, Jean-Emmanuel Hugonnet, Sophie Magnet, Michel Arthur, Jean-Luc Mainardi, Jure Stojan, Anamarija Zega
The D-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes...
September 2013: European Journal of Medicinal Chemistry
Elinor Meiby, Heather Simmonite, Loic le Strat, Ben Davis, Natalia Matassova, Jonathan D Moore, Michael Mrosek, James Murray, Roderick E Hubbard, Sten Ohlson
The increasing use of fragment-based lead discovery (FBLD) in industry as well as in academia creates a high demand for sensitive and reliable methods to detect the binding of fragments to act as starting points in drug discovery programs. Nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and X-ray crystallography are well-established methods for fragment finding, and thermal shift and fluorescence polarization (FP) assays are used to a lesser extent. Weak affinity chromatography (WAC) was recently introduced as a new technology for fragment screening...
July 16, 2013: Analytical Chemistry
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