RESEACH DIRECTION: DRUG DESIGN I
One common strategy for developing new drugs is large-scale combinatory
screening in which thousands of different molecules are tested for activity
changes. In addition, the availability of the human genome has led scientists
to investigate how to screen large numbers of proteins as possible drug
targets. Such high-throughput screening requires methodologies that are
readily performed in the laboratory and produce measurable parameters.
In addition, once possible drug candidates are identified, the methodology
must be amenable to distinguishing high-affinity, high-sensitivity drugs
from others that are less effective. For detailed biochemical studies, the
ability to perform the measurements over a versatile set of conditions would
also be required.
When a drug or ligand binds to a protein, heat is either released or
absorbed. Although these changes are small, the heat change can be meas-
ured accurately using calorimetry (calorimetry is a Latin-based word that
literally means the measurement of heat). There are several methods for
measuring heat in biochemical systems, with microcalorimeters being able
to measure thermodynamic para-
meters using volumes as small
as 0.1 mL. Differential scanning
calorimetery is typically used in
biochemistry to monitor folding
and unfolding of proteins. Measure-
ments of interactions between
molecules and proteins are typic-
ally performed using a technique
termed isothermal titration calori-
metry (ITC), which is performed at
constant temperature and involves
the systematic titration of the pro-
tein with the drug. Modern instru-
ments are able to determine the
energetics of drug binding with
high reliability and accuracy.
ITC measures the heat absorbed
or released when a drug is intro-
duced into a sample cell that con-
tains the protein and is allowed to
react (Figure 2.3). In a typical test
for a drug, a syringe contains a
concentrated solution of the drug
and small amounts of the drug are
injected in a stepwise manner into
a chamber containing the protein.
CHAPTER 2 FIRST LAW OF THERMODYNAMICS 27
ΔH Ka[D]/[P]totaldq/dtTimeqFigure 2.3A schematic diagram of an ITC sample chamber
and the results of a typical binding experiment. The sample
chamber contains the protein and the syringe has a
concentrated solution of the drug. At stepwise time intervals
a small volume of the drug is injected, and subsequent
bindingleads to the release of heat, q, at the rate dq/dt.
Modified from Freire (2004).