High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery

Pantoliano, Michael W.; Petrella, Eugene C.; Kwasnoski, Joseph D.; Lobanov, Victor S.; Myslik, James C.; Graf, E. R.; Carver, Ted; Asel, Eric; Springer, Barry A.; Lane, Pamela; Salemme, F.R.

doi:10.1089/108705701753364922

Cited by 258 publications

(359 citation statements)

References 41 publications

(55 reference statements)

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“…The calculated T m was higher than that in the absence of inhibitor, demonstrating how equilibrium binding ligands at concentrations higher than their K d values increase protein stability. Methods to estimate binding affinity from changes in protein thermal stability using a single concentration of ligand have been published previously (3)(4)(5)(6)(7)(8)(9)(10). A more accurate measure of the binding constant could be obtained by examining stability as a function of ligand concentration.…”

Section: Resultsmentioning

confidence: 99%

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Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using ThermoFluor

et al. 2005

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ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K b ) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T m can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30°C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T m . The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

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Section: Resultsmentioning

confidence: 99%

“…ThermoFluor measures the change in fluorescence of an environmentally sensitive dye on protein unfolding (9). These dyes are quenched in aqueous environments but have a large increase in quantum yield when bound to the hydrophobic interior of a protein.…”

mentioning

confidence: 99%

Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using ThermoFluor

et al. 2005

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“…56 Thermal unfolding of Rituximab was explored using a high-throughput microarray method, 57 in which the fluorescence is detected by a MyiQ single-color real-time PCR detection system (Bio-Rad Labs., Hercules, CA). A 480-nm excitation filter with 40-nm bandwidth and a 540-nm emission filter with 50-nm bandwidth were used.…”

Section: Sypro Orange Fluorescencementioning

confidence: 99%

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

et al. 2010

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Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lagtime behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size-exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.

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“…The thermal stability of GCase (Cerezyme) in the absence and presence of increasing concentrations of IFG was determined using differential scanning fluorimetry (DSF) by monitoring the increase in emission intensity of NanoOrange, [18][19][20]29] an environmentally sensitive fluorescent probe that undergoes fluorescence enhancement upon exposure to the hydrophobic interior of a protein. As the temperature is elevated, the fluorescence intensity of NanoOrange increases as a larger proportion of the GCase in solution unfolds, enabling binding of the probe to the hydrophobic interior.…”

Section: Ifg Enhances Global Stability and Activitymentioning

confidence: 99%

“…This network stabilized a particular active site conformation that the authors suggested promoted greater global stability. [7] Here we probe the effects of IFG-binding in solution on GCase global stability by differential scanning fluorimetry [18][19][20][21][22] and on local dynamics by amide hydrogen/ deuterium exchange coupled with proteolysis and mass spectrometry (H/D-Ex). [23][24][25][26][27][28] The ability to partially restore intracellular trafficking and lysosomal GCase localization of mutant forms of GCase was then inferred from activity assays performed on N370S/N370S and F213I/L444P patient fibro-blasts.…”

mentioning

confidence: 99%

Isofagomine Induced Stabilization of Glucocerebrosidase

Kornhaber¹,

Tropak

Maegawa

et al. 2008

ChemBioChem

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Structurally destabilizing mutations in acid β-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by ~15 °C and that it enhances mutant GCase activity in pretreated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/ deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions.

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High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery

Cited by 258 publications

References 41 publications

Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using ThermoFluor

Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using ThermoFluor

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

Isofagomine Induced Stabilization of Glucocerebrosidase

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