WHITEPAPER
Addressing the needs of drug discovery with
the MicroCal PEAQ-ITC instruments
Measurements and characterization of binding interactions between proteins and
low-molecular weight ligands are fundamental for drug discovery. Among the most
recognized challenges in characterizing binding interactions are (1) the need to
accurately assess a wide span of binding affinities (KD) and (2) accurately rank and
characterize low-molecular weight (LMW) ligands based on affinity, mechanism of
action, and energetics of interaction.
Reliable interpretation of binding data can be complicated by the presence of inactive
protein fraction or inaccurate assessment of protein concentration. Assessment of this
data can be further hampered by inherent uncertainty in concentration of compound
stocks. This uncertainty results from inaccurate measurement, limited solubility, or
potential chemical heterogeneity of the compounds such as presence of enantiomers
and isomers.
Isothermal titration calorimetry (ITC) directly measures heat released or absorbed
in a binding event, providing means for studying protein-small molecule interactions
in solution without the need for labeling or immobilization. A highly sensitive ITC
instrument and properly designed experimental conditions make it possible to account
for inaccurately assessed protein concentration or inactive protein fraction, and to
account for imprecise concentration of compound solutions. From quality control
(QC) to assay development and lead optimization, ITC has a role in improving
our understanding of biochemical data. Normalizing for protein or compound
concentrations and accounting for inactive populations of protein can improve decisionmaking processes, one example being a more careful assessment of changes in
enthalpic contributions to binding, which are critical in many best-in-class drug studies.
This white paper highlights the advantages of the new ITC instruments—MicroCal
PEAQ-ITC and MicroCal PEAQ-ITC Automated systems and MicroCal PEAQ-ITC
Analysis Software. The software allows analysis of binding interactions complicated
by the presence of inactive protein material, incorrect protein concentration, and/or
inaccurate concentration or heterogeneity of compound solutions.
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Improved SAR
Active protein concentration determination
ITC is utilized throughout early stage drug discovery, from assay optimization work and
secondary screening to late stages involving lead optimization. In addition, ITC can
be used in the protein purification QC step to assess batch variations and freeze-thaw
stability. In the earlier stage applications, one would typically test a set of compounds
including a well-established positive control. The periodic use of a positive control
ligand provides means for monitoring protein quality and establishing and confirming
active protein concentration throughout a series of ongoing ITC experiments. The
MicroCal PEAQ-ITC Analysis Software generates scatter plots of binding parameters
for a series of consecutive titrations. Such plots facilitate the detection of possible
trends and help to identify potential issues with assay setup and reagent quality.
Figure 1 presents a scatter plot of N values obtained for a series of consecutive ITC
titrations. It shows a trend in apparent number of binding sites on the target protein.
The gradual decrease in N value over time most probably reflects stability issues of
the target protein. Limited protein stability, batch-to-batch variation, and limited freezethaw stability of a target protein are common challenges associated with studies of
protein-ligand interactions. ITC data is useful to determine whether one lot of protein
differs in apparent activity (binding) relative to previous lots and provides potential
normalization criteria, which are useful across many assays run throughout the
screening/characterization process.
Figure 1: Scatter plot of N-values generated by MicroCal PEAQ-ITC software for a series of consecutive
titrations of 5 µM bovine carbonic anhydrase II (bCAII) with 50 µM ethoxzolamide in phosphate-buffered saline
(PBS), 25°C. The experiments were conducted in the MicroCal ITC Automated system (at a time interval of 1.5
h between experiments).
With the concentration of the active protein established and used in the ITC data
analysis, any remaining errors in the apparent stoichiometry (i.e. non integer values 1,2
…etc) can be assigned to incorrect concentration determination of the ligand.
Accurate ligand concentration determination
For many reasons, it is not uncommon in drug discovery that the concentration of
compound solutions is given with significant errors. This inaccuracy directly affects
the quality of the binding parameters returned by fitting binding data; most notably
in the correct determination of the enthalpic and entropic contributions to binding. In
the new MicroCal PEAQ-ITC Analysis Software, it is possible to identify and account
for the error in the concentration of the compound to minimize any errors in the
thermodynamic measurements.
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Addressing the needs of drug discovery with the MicroCal PEAQ-ITC instruments
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Figure 2 presents the scatter in the stoichiometry (N) determined by ITC for an
interaction of a target protein with a series of LMW hits in a drug discovery and
development project. This was conducted by a client using the MicroCal Auto iTC200.
The N values ranged from 0.2 to 1.8 and were not correlated with a fraction of inactive
protein which was already established using the method described above. In addition,
1:1 binding stoichiometry was expected for the compounds based on X-ray structures
of the complexes. It was clear therefore that differences in the apparent stoichiometry
were due to incorrect determination of the ligand concentrations.
Figure 2: Case study (client-provided data). Summary of N values obtain for interactions of a target protein with
a series of LMW hits in a drug discovery and development project conducted on MicroCal Auto iTC200.
These errors directly impact on the enthalpy data generated by the ITC making it very
difficult to interpret the differences in the thermodynamics of interaction of each ligand
to the target protein.
The new MicroCal PEAQ-ITC Analysis Software can account for this by allowing the
concentration of the LMW ligand in the syringe to vary while setting stoichiometry
value to 1 in the fitting process (this can be performed automatically on many data
sets simultaneously). Figure 3 shows the enthalpy data returned with and without this
correction factor.
Figure 3: Comparison of the enthalpy data returned for the interaction of a series of LMW hits with a target
protein in a drug discovery program, with (light blue) and without (dark blue) the correction for ligand
concentration. The compounds are listed in order of descending affinity.
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Addressing the needs of drug discovery with the MicroCal PEAQ-ITC instruments
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It is clear that the enthalpy data returned is dramatically different when the errors
in ligand concentration are factored into the analysis and clearly will have a large
impact on the interpretation of the SAR (structure activity relationships) of this lead
optimization study.
Another consequence of inaccurate ligand concentrations is that experiments may be
difficult to analyze due to the incomplete nature of the binding isotherm and may need
repeating. An example of this is shown in Figure 4.
Figure 4: Examples of ITC data for the titration of a LMW ligand into a protein where the ligand concentration
was in significant error. Raw ITC data (upper panels) and integrated data fitted to One-set-of-sites interaction
model (lower panels). The fit was conducted (A) without and (B) with accounting for error in compound
concentration. The insets in the lower panel contain the fitted parameters returned by the analysis software.
The MicroCal PEAQ-ITC Analysis Software can fit these incomplete binding isotherms
by using a constant offset, representing the control heats, in the fitting process. By
combining the control offset and varying the ligand concentration in the fitting process,
previously ‘unfittable’ or difficult data sets can be analyzed automatically in a nonsubjective manner. In many cases this is sufficient for the application and, at least,
can be used to establish the correct concentration of the compound stock. In this
example, the ligand concentration was initially estimated to be 200 µM but was found
to be 126 µM. This error had a large impact on the enthalpy (ΔH) and entropy (-TΔS)
which changed by -2.8 kcal/mole and 2.28 kcal/mol, respectively, while the apparent
KD changed from 241 nM to 122 nM. These differences highlight the value of the
new features in the analysis software and the benefits it will bring to robust data
interpretation.
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Addressing the needs of drug discovery with the MicroCal PEAQ-ITC instruments
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Simultaneous affinity determination for isomers
and enantiomers
In late-stage drug discovery, a significant number of compounds are synthesized as
mixtures of enantiomers or isomers. In many of these cases, the compound mixture
can be separated by high-performance liquid chromatography (HPLC) and one or
more isomers can be tested individually. However in some cases, particularly those
involving mixtures of enantiomers, separation can be difficult and time-consuming,
and in these cases the mixtures must be tested directly. Biochemical assays primarily
provide information on the tightest binding component in the mixture. In contrast,
ITC can provide binding information about both the strong and the weaker binder
simultaneously in a single experiment.
The biphasic binding isotherm in Figure 5 represents an example how the ITC data
may look for the injection of an enantiomeric ligand mixture into a target protein
in the cell. Similarly, biphasic isotherms are observed when high affinity ligands
are intentionally mixed with weaker ligands as a form of competition experiment
to determine KDs outside the range of direct measurements.This differs from the
more typical approach of injecting the tight binding ligand into a mixture of the target
and the weak inhibitor but has the advantage that both inhibitors can be resolved
simultaneously and without prior knowledge of the interaction parameters of the weak
interaction.
Figure 5: MicroCal iTC200 data for the titration of enantiomeric compound into target protein (client-provided
data). The binding isotherm is clearly biphasic; however, analysis of the data with Origin ITC data analysis was
not straightforward.
To demonstrate the utility of the MicroCal PEAQ-ITC Analysis Software for the analysis
of complex binding isotherms we have performed experiments with mixed ligands
(ethoxzolamide (EZA), and furosemide (FUR)) in the syringe injected into a target
protein (Bovine carbonic anhydrase II, bCAII) in the cell. The data are shown in figure
6 and represent the type of biphasic isotherm data that is expected for an enantiomeric
mixture or a ‘syringe’ competition experiment. 5
Addressing the needs of drug discovery with the MicroCal PEAQ-ITC instruments
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Figure 6: Raw and normalized heat plots for the titrations of the 1:1 mixture of EZA and FUR into bCAII.
The titrations were carried out at 160 µM total concentration of the compounds in the syringe and 10 µM
concentation of bCAII in the cell (left) or at 100 µM concentration of the compound mixture and 5 µM
concentation of bCAII in the cell (right). 18 × 2 µl injection were made and the buffer was PBS, 2 v/v % DMSO at
25°C.
These experiments demonstrate the level of resolution that can be achieved using
the MicroCal PEAQ-ITC and the potential for quantitative characterization of the
binding of mixtures. The fitting of complex ITC data, and in particular the fitting of
these competitive experiments, has been made simpler in the MicroCal PEAQ-ITC
software than in analysis software previously available on earlier models. While the
input of good initial guesses into the fitting software is always a good start, the current
software contains more appropriate numerical boundaries increasing the chances
of a successful fit and minimizing the risk of the fitting process getting trapped in a
local minima. Fitting these data also benefited from combining the Simplex fitting and
Marquardt-Levenberg fitting approaches available in the software. More specifically,
using the Simplex approach at the start and finalizing using the Marquardt-Levenberg
fitting algorithm. The thermodynamic parameters obtained using this approach with the
‘two-sites’ model and ‘ligand in cell’ setting are summarized in Table 1.
Table 2: Results of the analysis of the data collected for titrations of the 1:1 mixtures of
EZA and FUR inhibitors into bCAII with a new MicroCal PEAQ-ITC Analysis Software.
For comparison, binding parameters established in separate titrations (n = 3) of the
compounds were: for EZA KD= 1.3 ± 1.0 nM, enthalpy ΔH = -15.10 ± 0.01 kcal/mol; for
FUR KD = 360 ± 70 nM, ΔH = -6.30 ± 0.02 kcal/mol. All titrations were conducted on
the new MicroCal PEAQ-ITC system in PBS and 2% v/v DMSO, at 25°C. The binding
parameters are reported along with the standard errors given in brackets.
bCAII in
cell, mM
N1 (sites)
KD1 (nM)
DH1 (kcal/
N2 (sites)
KD2 (nM)
mol)
DH1 (kcal/
mol)
5
0.5 (4)
0.8 (100)
-15.1 (1)
0.6 (7)
700 (43)
-7 (13)
10
0.49 (3)
1 (93)
-15.2 (2)
0.56 (3)
630 (42)
-7.1 (5)
The data compare well with the thermodynamic values obtained for these interactions
when measured independently (see table 1 legend).
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Addressing the needs of drug discovery with the MicroCal PEAQ-ITC instruments
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Summary and conclusions
The new MicroCal PEAQ-ITC instrument along with improved signal stability, mixing
and signal-to-noise characteristics has data analysis software well-suited for use in the
biophysical laboratories involved in small-molecule drug discovery.
The analysis is completely automated, minimizing user subjectivity in assessing data
quality and the analysis process. Data quality assessment and fitting is performed
rapidly allowing for analysis of large data sets of 50 or more experiments in seconds.
Key additional features of the new MicroCal PEAQ-ITC Analysis Software package
allow for the determination of the active concentration of the target protein or the ligand
concentration or both when the appropriate controls are used. This provides for the
more accurate determination of the affinity and thermodynamic allowing for rigorous
structure-activity relationship in hit validation and lead optimization programs.
In addition, the new ITC data analysis software has tools that simplify the fitting of
complex binding isotherms that may be observed when titrating with enantiomeric
mixtures, some competition experiments or with targets with more than one binding
sites.
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Addressing the needs of drug discovery with the MicroCal PEAQ-ITC instruments
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