Related ArticlesALARM NMR: a rapid and robust experimental method to detect reactive false positives in biochemical screens.
J Am Chem Soc. 2005 Jan 12;127(1):217-24
Authors: Huth JR, Mendoza R, Olejniczak ET, Johnson RW, Cothron DA, Liu Y, Lerner CG, Chen J, Hajduk PJ
High-throughput screening (HTS) of large compound collections typically results in numerous small molecule hits that must be carefully evaluated to identify valid drug leads. Although several filtering mechanisms and other tools exist that can assist the chemist in this process, it is often the case that costly synthetic resources are expended in pursuing false positives. We report here a rapid and reliable NMR-based method for identifying reactive false positives including those that oxidize or alkylate a protein target. Importantly, the reactive species need not be the parent compound, as both reactive impurities and breakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactive molecules by nuclear magnetic resonance) and is based on monitoring DTT-dependent (13)C chemical shift changes of the human La antigen in the presence of a test compound or mixture. Extensive validation has been performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity. This included comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing a collection of more than 3500 compounds containing HTS hits from 23 drug targets. The data show that current in silico filtering tools fail to identify more than half of the compounds that can act via reactive mechanisms. Significantly, we show how ALARM NMR data has been critical in identifying reactive compounds that would otherwise have been prioritized for lead optimization. In addition, a new filtering tool has been developed on the basis of the ALARM NMR data that can augment current in silico programs for identifying nuisance compounds and improving the process of hit triage.
[NMR paper] A general NMR method for rapid, efficient, and reliable biochemical screening.
A general NMR method for rapid, efficient, and reliable biochemical screening.
Related Articles A general NMR method for rapid, efficient, and reliable biochemical screening.
J Am Chem Soc. 2003 Nov 26;125(47):14620-5
Authors: Dalvit C, Ardini E, Flocco M, Fogliatto GP, Mongelli N, Veronesi M
High-throughput screening is usually the method of drug-lead discovery. It is now well accepted that, for a functional assay, quality is more important than quantity. The ligand-based or protein-based NMR screening methodologies for detecting compounds...
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11-24-2010 09:16 PM
[NMR paper] Microdrop screening: a rapid method to optimize solvent conditions for NMR spectrosco
Microdrop screening: a rapid method to optimize solvent conditions for NMR spectroscopy of proteins.
Related Articles Microdrop screening: a rapid method to optimize solvent conditions for NMR spectroscopy of proteins.
J Biomol NMR. 1998 Nov;12(4):493-9
Authors: Lepre CA, Moore JM
Determining appropriate solvent conditions is a crucial first step for carrying out NMR spectroscopy of proteins, but rapid and efficient methods for doing so are currently lacking. Microdrop screening examines a large number of different solvent conditions using...
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[NMR paper] Internal mobility of reactive-site-hydrolyzed recombinant Cucurbita maxima trypsin in
Internal mobility of reactive-site-hydrolyzed recombinant Cucurbita maxima trypsin inhibitor-V characterized by NMR spectroscopy: evidence for differential stabilization of newly formed C- and N-termini.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles Internal mobility of reactive-site-hydrolyzed recombinant Cucurbita maxima trypsin inhibitor-V characterized by NMR spectroscopy: evidence for differential stabilization of newly formed C- and N-termini.
Biochemistry. 1996 Sep 24;35(38):12503-10
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[NMR paper] The use of 19F NMR in the study of protein alkylation by fluorinated reactive interme
The use of 19F NMR in the study of protein alkylation by fluorinated reactive intermediates.
Related Articles The use of 19F NMR in the study of protein alkylation by fluorinated reactive intermediates.
Adv Exp Med Biol. 1991;283:735-8
Authors: Harris JW, Anders MW
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08-21-2010 11:16 PM
Robust structure-based resonance assignment for functional protein studies by NMR
Abstract High-throughput functional protein NMR studies, like protein interactions or dynamics, require an automated approach for the assignment of the protein backbone. With the availability of a growing number of protein 3D structures, a new class of automated approaches, called structure-based assignment, has been developed quite recently. Structure-based approaches use primarily NMR input data that are not based on J-coupling and for which connections between residues are not limited by through bonds magnetization transfer efficiency. We present here a robust structure-based assignment...
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HIFI-C: a robust and fast method for determining NMR couplings from adaptive 3D to 2D projections
HIFI-C: a robust and fast method for determining NMR couplings from adaptive 3D to 2D projections
Gabriel Cornilescu, Arash Bahrami, Marco Tonelli, John L. Markley and Hamid R. Eghbalnia
Journal of Biomolecular NMR; 2007; 38(4); pp 341-351
Abstract:
We describe a novel method for the robust, rapid, and reliable determination of J couplings in multi-dimensional NMR coupling data, including small couplings from larger proteins. The method, “High-resolution Iterative Frequency Identification of Couplings” (HIFI-C) is an extension of the adaptive and intelligent data collection approach...
ALARM NMR: a rapid and robust experimental method to detect reactive false positives
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