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Isothermal TitrationCalorimetry and Drug Design
In structure-based drug design (SBDD), full structural knowl-
edge of the protein target molecule provides information on how
a potential drug interacts with the target. Computer modeling
algorithms and molecular simulations are used to predict quanti-
tative structure-activity relationships (QSAR) and these data are
used to optimize the design of new drugs. However, molecular
modeling is limited by a lack of experimental thermodynamic
data to verify these models.
Ultrasensitive Isothermal Titration Calorimetry (ITC) providescomplete thermodynamic characterization of the binding of adrug to its therapeutic target, including enthalpy (ΔH) and
FIGURE 1. Thermodynamic signatures for three different drugs binding tosame target. Scheme A: good hydrogen bonding and a conformational
entropy (TΔS), free energy (ΔG) and binding affinity (KB). These
change. Scheme B: binding dominated by hydrophobic interactions.
thermodynamics parameters are related to each other by the fol-
Scheme C: Favorable hydrogen bonding and hydrophobic interactions.
susceptibility to mutations that can cause drug resistance.
Scheme C has favorable ΔH and ΔS, the most favorable thermo-
For spontaneous reactions, ΔG is negative, and ΔG is directly
Effective drugs are expected to have high binding affinity and
related to the binding affinity. The tighter the binding, the more
selectivity for target. Drugs for infectious diseases also should be
negative the ΔG. Enthalpy and entropy both contribute to ΔG.
adaptive, and bind to a broad spectrum of targets, such as protein
ΔH reflects the strength of the drug-
mutants and families of targets. ITC is utilized for the following:
target interaction relative to those with solvent, primarily due to
• Optimization of binding affinity:
ITC determines thermody-
hydrogen bond formation, and van der Waals interactions. WhenΔ
namic signatures of lead compounds binding to target. Choose
H is negative, binding is enthalpically favored. Favorable
lead compound(s) with favorable enthalpy and further optimize
enthalpy requires correct placement of hydrogen bond acceptor
them by the addition of hydrophobic regions to the core drug.
and donor groups at the binding interface.
Drugs with high binding affinities have lower dosage require-
A positive TΔS results in entropically
ments and consequently fewer side effects.
favored binding. Favorable entropy changes are primarily due to
• Binding adaptability:
Design drugs which can bind to mutant
hydrophobic interactions, due to an increase in solvent entropy
forms of target proteins, thereby reducing drug resistance. Also
from burial of hydrophobic groups and release of water upon
design drugs which bind to a family of related targets.
binding, as well as minimal loss of conformational degrees offreedom.
• Improvement of binding selectivity:
Design drugs which bind
selectively to a specific target, bind less selectively to serum
Figure 1 shows different thermodynamic signatures that might be
proteins or other non-specific targets.
observed for three different drugs binding to the same target.
Each drug has identical ΔG and binding affinity for the target.
Scheme A has favorable ΔH, characteristic of hydrogen bondformation, and an unfavorable TΔS. Drugs showing this bindingprofile typically have large degree of flexibility, as well as highpolarity, which could cause problems with membrane permeabil-ity in vivo
. Scheme B has a favorable TΔS, indicating that bind-ing is driven by hydrophobic interactions, and an unfavorableΔH. Drugs showing this binding profile are very hydrophobicand are poorly soluble, and there are also conformationalrestraints leading to lack of adaptability and consequently a high
Ultrasensitive Calorimetry for the Life SciencesTM
Optimization of Binding Affinity
In typical lead optimization, compounds are initially screenedfor binding affinity to target, with no information about thermo-
One goal in drug design is to make drugs which bind to their tar-
dynamics. Compounds with high affinity continue through the
get with the highest binding affinity. Higher affinity results in
discovery pipeline, but may not lead to a successful drug.
lower dosage requirement, greater specificity, better drug effica-
Freire’s research indicates that a better strategy is identifying
cy, reduced side effects, and less drug resistance. Research by
lead compounds with highly favorable ΔH, and optimizing these
Ernesto Freire’s group at Johns Hopkins University has shown
drugs by addition of hydrophobic groups. This could result in a
that thermodynamics from ITC data can be used to characterize
highly-specific drug with tight binding affinity. Preliminary work
HIV-1 protease inhibitors, and binding affinity is optimized by
has been done on the design of SARS drugs using thermody-
overcoming enthalpy-entropy compensation.1-5
namic signatures.6 The first generation inhibitors of SARS 3CLproprotease have enthalpically favorable binding, and these drugs
Figure 2 shows the thermodynamic signatures of binding of sev-
are being optimized to increase binding affinity.
eral inhibitors to wild-type HIV-1 protease. Binding of indinavir,nelfinavir, and saquinavir are driven by entropy, due to
This ITC-based QSAR has also been used to develop new drugs
hydrophobic interactions between the drug and the target.
which bind to DNA gyrase, by researchers at AstraZeneca.7 They
Binding of these inhibitors have unfavorable enthalpy. Ritonavir,
observed that triazine inhibitors of DNA gyrase tend to have
amprenivir, lopinavir, and two experimental inhibitors KNI-272
similar binding energetics, and they looked for triazine deriva-
and KNI-764 have favorable binding enthalpy, as well as a favor-
tives which had a markedly different thermodynamic signature,
able entropic contribution. Binding interactions with these
especially in ΔH, and studied these different derivatives for their
inhibitors have both favorable hydrogen bond formation as well
structural differences and binding modes. This kind of screening
as hydrophobic interactions. These inhibitors are second and
can be useful in the discovery of new drugs.
third generation inhibitors. Loprinavir has the highest bindingaffinity of these inhibitors (KB of 1.2 x 1011 M-1), measured by
Drug resistance is a serious side effect associated with antiviral therapies, due to the appearance of viral strains withmutant forms of target protein. Mutants with reduced bindingaffinity for inhibitor typically maintain affinity for substrate.
Freire’s group has studied the thermodynamics of inhibitor bind-ing to mutant strains of HIV-1 protease.3-5 One multi-drug resis-tant mutant with six amino acid mutations of HIV-1 protease(MDR-HM) had binding affinity for inhibitor 2 to 3 orders ofmagnitude lower than wild-type.5 Study of these mutationsdemonstrate individual effects of each individual mutation, aswell as cooperative interactions between distal mutations.
The determination of enthalpic and entropic components of thedrug resistant HIV-1 protease mutants relative to wild type,
FIGURE 2. Thermodynamic signatures of binding of inhibitors to wild-type
along with structural studies, was used to develop guidelines in
HIV-1 protease. Indinavir, nelfinavir, saquinavir, ritonavir,amprenavir, and
the design of inhibitors more adaptable to drug target mutations.5
lopinavir are in clinical use. KNI-272 and KNI-764 are experimental protease
inhibitors. Data from References 4 and 5. Refer to Reference 5 for design ofITC experiments.
• Maximize ΔH contribution to ΔG for enthalpically favorable
Hydrogen bond formation and favorable van der Waals interac-tions between drug and target results in favorable enthalpy.
• Design drug-target interactions with highest binding affinity
Hydrogen bond formation is a result of optimal placement of
and specific hydrogen bonding between core of the drug and
hydrogen bond donor and acceptor groups on the drug and tar-
highly-conserved region of the target’s binding site.
get, and is highly directional and specific. Hydrogen bond
• Introduce non-constrained functional groups on inhibitor facing
donors and receptors may be in close proximity in a crystal
mutation-prone regions of binding site of target. Since this will
structure but may have little effect on binding affinity, due to
make ΔS of binding less favorable by loss of conformational
degrees of freedom of inhibitor, inhibitor ΔH must be highly
A common strategy in drug design is the addition of non-polar
favorable to maintain high affinity.
groups to increase hydrophobic interactions between target andcompound. However, hydrophobic interactions are non-specificcompared to hydrogen bonding. Freire’s research demonstratesthat drugs with favorable enthalphy AND entropy have highestaffinity, due to specific hydrogen bond formation and van derWaals interactions as well as hydrophobic interactions with thetarget.
Thermodynamics and ITC have also been used to design adap-
tive inhibitors. This is achieved by designing an inhibitor with atight and specific binding interaction with the conserved region
1. Velazquez-Campoy, A., Todd, M.J., Freire, E. (2000) HIV-1 protease
of the binding site of the primary target, and introduces flexible
inhibitors: Enthalpic versus entropic optimization of the binding
affinity. Biochemistry 39
asymmetric functional groups to be able to bind the other relatedtargets. This approach has been used to design anti-malarial
2. Velazquez-Campoy, A., Kiso, Y., Freire, E. (2001) The binding ener-
drugs against proteases of Plasmodium falciparum
, plasmepsin I,
getics of first- and second-generation HIV-1 protease inhibitors:
II, IV, and histo-asparyl protease (HAP).8,9 A tight-binding
Implications for drug design. Arch. Biochem. Biophys. 390
inhibitor to plasmepsin II also inhibited secondary targets, due to
3. Velazquez-Campoy, A., Vega, S., Freire, E. (2002) Amplification of
the adaptive group on the inhibitor.
the effects of drug resistance mutations by background polymor-
phisms in HIV-1 protease from African subtypes. Biochemistry 41
4. Ohtaka, H., Velazquez-Campoy, A., Xie, D., Freire, E. (2002)
Overcoming drug resistance in HIV-1 chemotherapy: The binding
Binding selectivity is dependent on the affinity of drug to both
thermodynamics of amprenavir and TMC-126 to wild-type and
desired and unwanted targets, as well as the KB ratio of the two
drug-resistant mutants of the HIV-1 protease. Protein Sci
binding events. Drugs can bind to non-specific targets, such as
serum proteins, or to other target proteins with different bindingaffinities. When a drug binds serum proteins, the drug’s bioavail-
5. Ohtaka, H., Schön, A., Freire, E. (2003) Multidrug resistance to
HIV-1 protease inhibition requires cooperative coupling between
ablity and efficacy are reduced, requiring a higher dosage. ITC
distal mutations. Biochemistry 42
has been used to study the binding of HIV-1 protease inhibitorsto human serum proteins.10 HIV-1 protease inhibitors had a sig-
6. Bacha, U.M., Barrila, J.A., Velazquez-Campoy, A., Leavitt, S.,
nificant binding affinity for α1-acid glycoprotein (AAG) and a
Freire, E. (2004) Development of potent inhibitors of the SARS
relatively low binding affinity for human serum albumin (HSA).
associated coronavirus protease 3CLpro. Biophys J. 86
, 97a. (Poster at
Using AAG and HSA concentration simulating in vivo
tions, up to 10 times higher concentration was needed to inhibit
7. Ward, W.H.J., Holdgate, G.A. (2001) Isothermal titration calorime-
HIV-1 protease relative to the amount needed to inhibit in
try in drug discovery. Prog. Med. Chem
absence of serum proteins. When drug-resistant HIV-1 protease
8. Nezami, A., Luque, I., Kimura, T., Kiso, Y., Freire, E. (2002)
mutants were used, up to 2000-fold more inhibitor was required
Identification and characterization of allophenylnorstatime-based
to inhibit protease, and this is beyond the solubility limit of the
inhibitors of plasmepsin II, and anti-malarial target. Biochemistry
ITC and structural studies can be used to develop new drugs to
9. Nezami, A., Kimura, T., Hidaka, K., Kiso, A., Liu, J., Kiso, Y.,
maximize selectivity and specific binding to intended target by
Goldberg, D.E., Freire, E. (2003) High-affinity inhibition of a family
optimization of enthalpy and entropy.
of Plasmodium falciparum
protease by a designed adaptive inhibitor.Biochemistry 42
10. Schön, A., Ingaramo, M.d.M., Freire, E. (2003) The binding of HIV-
1 protease inhibitors to human serum proteins. Biophys. Chem. 105
ITC studies on HIV-1 protease inhibitors, anti-malarial drugs,SARS drugs, and DNA gyrase inhibitors have generated thermo-dynamic signatures of drug-target interactions. These data, usedwith structural analysis, provide valuable information in thedesign of new drugs which bind with higher affinity and selectiv-ity, as well as adaptability. The introduction of ITC instrumenta-tion with increased throughput allows use of microcalorimetryearlier in drug discovery pipelines.
Acknowledgement: MicroCal, LLC thanks Dr. Ernesto Freire forreviewing this application note.
Ultrasensitive Calorimetry for the Life SciencesTM
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