et al. screened the same 930-compound fragment library against BRD4 using both
1 H CPMG and PrOF NMR to investigate the correlation between the ligand- and
protein-observed methods [94]. An 85% assay overlap was found for PrOF NMR
and
1 H CPMG NMR competitive hits. This result indicates that under the assay
conditions used, the assays show a good correlation. Additionally, the authors
showed rank ordering of hits via DSF and ITC is consistent with rank ordering of
hits by PrOF NMR. Finally, K d values determined by PrOF NMR and ITC were
compared. A variation of 1.4-4.7 fold in K d value indicates a good correlation
between the assays for K d determination.
4.2.6 ITC Can Determine the Thermodynamics and Stoichiometries
of Bromodomain Complexes
Isothermal titration calorimetry (ITC) is a gold standard for accurately determining
all of the thermodynamic binding properties including the K d , enthalpy, entropy, and
the complex stoichiometry. For example, Filippakopoulos et al. used ITC to determine the affinity and stoichiometry of a variety of bromodomains binding to multiacetylated peptide histone mimics [20]. The ligand of interest is titrated into a
constant concentration of protein (or vice versa). The heat of the binding interaction
is measured as the amount of energy used to keep the reaction at a constant
temperature. ITC is time-consuming and requires a high concentration of ligand
based on a need for a large molar excess above the protein concentration. Although
ITC is not ideal for bromodomain screening, ITC is a sensitive technique that does
not require modification to the ligand or protein of interest and can accurately
measure the K d of strong and weak binding compounds. This method is often used
to characterize hits and fully elaborated chemical inhibitors and probes. Additional
label-free methods for determining K d include bio-layer interferometry and microscale thermophoresis and surface plasmon resonance (SPR).
4.2.7 Kinetic Analysis of Interactions Using SPR
Surface plasmon resonance (SPR) is a direct-binding assay which has been used
to screen small-molecule libraries against multiple families of bromodomains
[104]. SPR measures the refractive index of a gold-plated surface called a chip.
The principle of the assay is that a single wavelength laser is directed at a surface that
reflects the light back at a detector. To measure binding affinities, the surface is first
immobilized with the protein of interest. If a binding partner is passed over the
protein and binds, the effective mass of the protein increases, altering the refractive
index of the chip, and causes a change in signal. Data collected via SPR are
displayed as sensograms (Fig. 15). Since this assay measures real-time interactions,
the full kinetics (k on and k off rates) of the ligand can be determined. If the ligand is
measured at several concentrations, the binding sensograms can be plotted as a
function of concentration, and a K d can be determined. This can be done relatively
316
W. C. K. Pomerantz et al.
1 H CPMG and PrOF NMR to investigate the correlation between the ligand- and
protein-observed methods [94]. An 85% assay overlap was found for PrOF NMR
and
1 H CPMG NMR competitive hits. This result indicates that under the assay
conditions used, the assays show a good correlation. Additionally, the authors
showed rank ordering of hits via DSF and ITC is consistent with rank ordering of
hits by PrOF NMR. Finally, K d values determined by PrOF NMR and ITC were
compared. A variation of 1.4-4.7 fold in K d value indicates a good correlation
between the assays for K d determination.
4.2.6 ITC Can Determine the Thermodynamics and Stoichiometries
of Bromodomain Complexes
Isothermal titration calorimetry (ITC) is a gold standard for accurately determining
all of the thermodynamic binding properties including the K d , enthalpy, entropy, and
the complex stoichiometry. For example, Filippakopoulos et al. used ITC to determine the affinity and stoichiometry of a variety of bromodomains binding to multiacetylated peptide histone mimics [20]. The ligand of interest is titrated into a
constant concentration of protein (or vice versa). The heat of the binding interaction
is measured as the amount of energy used to keep the reaction at a constant
temperature. ITC is time-consuming and requires a high concentration of ligand
based on a need for a large molar excess above the protein concentration. Although
ITC is not ideal for bromodomain screening, ITC is a sensitive technique that does
not require modification to the ligand or protein of interest and can accurately
measure the K d of strong and weak binding compounds. This method is often used
to characterize hits and fully elaborated chemical inhibitors and probes. Additional
label-free methods for determining K d include bio-layer interferometry and microscale thermophoresis and surface plasmon resonance (SPR).
4.2.7 Kinetic Analysis of Interactions Using SPR
Surface plasmon resonance (SPR) is a direct-binding assay which has been used
to screen small-molecule libraries against multiple families of bromodomains
[104]. SPR measures the refractive index of a gold-plated surface called a chip.
The principle of the assay is that a single wavelength laser is directed at a surface that
reflects the light back at a detector. To measure binding affinities, the surface is first
immobilized with the protein of interest. If a binding partner is passed over the
protein and binds, the effective mass of the protein increases, altering the refractive
index of the chip, and causes a change in signal. Data collected via SPR are
displayed as sensograms (Fig. 15). Since this assay measures real-time interactions,
the full kinetics (k on and k off rates) of the ligand can be determined. If the ligand is
measured at several concentrations, the binding sensograms can be plotted as a
function of concentration, and a K d can be determined. This can be done relatively
316
W. C. K. Pomerantz et al.