Assessing compound binding to the Eg5 motor domain using a thermal shift assay

McDonnell, Patricia A.; Yanchunas, Joseph; Newitt, John A.; Tao, Liyuan; Kiefer, Susan E.; Ortega, Marie; Kut, S. A.; Burford, Neil T.; Goldfarb, Valentina; Duke, Gerald J.; Shen, Henry; Metzler, William J.; Doyle, Michael L.; Chen, Zhong; Tarby, Christine M.; Borzilleri, R. M.; Vaccaro, Wayne; Gottardis, Marco M.; Lu, Songfeng; Crews, Donald; Kim, Kyoung; Lombardo, Louis J.; Roussell, Deborah L.

doi:10.1016/j.ab.2009.05.044

Cited by 19 publications

(17 citation statements)

References 44 publications

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“…The majority of the fragments, 73.2% (915 in total), displayed negative ΔT m values. This could be attributed to fragments stabilizing the unfolded state of the protein, or fragments aggregating and causing early destabilization and unfolding of the protein (37). Some fragments that possessed scaffolds identified as hits in this primary screen were members of this group displaying negative ΔT m values.…”

Section: Resultsmentioning

confidence: 99%

Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery

Silvestre

Blundell

Abell

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

100

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In fragment-based drug discovery, the weak affinities exhibited by fragments pose significant challenges for screening. Biophysical techniques are used to address this challenge, but there is no clear consensus on which cascade of methods is best suited to identify fragment hits that ultimately translate into bound X-ray structures and provide bona fide starting points for synthesis. We have benchmarked an integrated biophysical approach for fragment screening and validation against Mycobacterium tuberculosis pantothenate synthetase. A primary screen of 1,250 fragments library was performed by thermal shift, followed by secondary screen using one-dimensional NMR spectroscopy (water ligand observed gradient spectroscopy and saturation transfer difference binding experiments) and ultimate hit validation by isothermal titration calorimetry and X-ray crystallography. Our multibiophysical approach identified three distinct binding sites for fragments and laid a solid foundation for successful structure-based elaboration into potent inhibitors.drug design | hot spots | protein-ligand interactions

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Section: Resultsmentioning

confidence: 99%

Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery

Silvestre

Blundell

Abell

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

100

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“…22 As shown in Table 1, these three series of dihydropyrazolo [3,4-b]pyridine compounds were mostly only moderately to weakly potent growth inhibition of HCT116 cell line, while compound 3c and 3d was found to have more potency against HCT116 cell line with the IC 50 values of 4.97 and 2.36 lM, respectively, compared with that of the positive control CPUYL064's 12.70 lM. Compounds with thienyl group at the R 1 group, such as 4b, 4c and 4d, showed moderate anti-proliferative cytotoxic activities with an IC 50 in the 11-25 lM range.…”

Section: Resultsmentioning

confidence: 97%

Design, synthesis and bioevaluation of dihydropyrazolo[3,4-b]pyridine and benzo[4,5]imidazo[1,2-a]pyrimidine compounds as dual KSP and Aurora-A kinase inhibitors for anti-cancer agents

You

Yang

et al. 2010

Bioorganic & Medicinal Chemistry

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“…however, several communications describe combined analyses of potency versus thermal stability. 12,14,15 Very interestingly, in their study on the fatty acid amide hydrolase, slaymaker et al 15 were able to identify two different compound populations once they plotted a graph lnK i versus t m . unfortunately, no thermodynamic measurements were realized with these compounds.…”

Section: Resultsmentioning

confidence: 99%

“…the binding of a ligand to a protein leads to the formation of a complex protein-ligand that usually exhibits a higher melting temperature, t m b , than the free protein. experiments have shown a good correlation between ligand potency measured in biochemical assays and shift in melting temperatures (Δt m = t m b -t m ) estimated in ftsa, [12][13][14][15] and ftsa is already routinely used to triage hits identified from high-throughput screening (hts) campaigns. moreover, Δt m s obtained from ftsa are directly connected to the thermodynamic parameters of the ligand-protein interaction, 7,16,17 providing a link to the thermodynamic signatures of the compounds.…”

mentioning

confidence: 99%

Leveraging the Contribution of Thermodynamics in Drug Discovery with the Help of Fluorescence-Based Thermal Shift Assays

et al. 2011

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The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.

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Assessing compound binding to the Eg5 motor domain using a thermal shift assay

Cited by 19 publications

References 44 publications

Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery

Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery

Design, synthesis and bioevaluation of dihydropyrazolo[3,4-b]pyridine and benzo[4,5]imidazo[1,2-a]pyrimidine compounds as dual KSP and Aurora-A kinase inhibitors for anti-cancer agents

Leveraging the Contribution of Thermodynamics in Drug Discovery with the Help of Fluorescence-Based Thermal Shift Assays

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