all sites, i.e.any sequence dependence for drug binding is ignored, and nthe binding site size, i.e.the number
of base pairs occupied by a bound drug. The neighbour exclusion model predicts that binding isotherms
plotted in the form r/Cfversus r should be initially linear but should curve asymptotically as values of r/Cf
approach zero. This is exactly what is observed in experiments, and the neighbour exclusion model can
describe accurately the binding of intercalators to DNA.^22
However, it is common for non-integral numbers for the parameter nto emerge from this analysis, which
implies that the neighbour exclusion model is not adequate to describe the experimental data. Obvious rea-
sons for this are that certain assumptions are not appropriate for the particular system under study. In such
cases, more complex versions of the model that account for sequence specificity or the influence of
co-operativity are required (Section 9.9).
9.6.4 Apportioning the Free Energy for DNA Intercalation Reactions
The likelihood of success in rational DNA-binding drug design programmes would be greatly enhanced if,
in addition to structural studies, thermodynamic analyses were to be carried out for a wide range of ligand–
DNA complexes. Determination of the free energy change for binding is useful but insufficient. As a min-
imum, the overall observed free energy should be examined in terms of component enthalpic and entropic
terms. Modern advances in titration microcalorimetry have given experimentalists the ability to under-
take novel and detailed thermodynamic investigations into ligand–DNA interactions. These tools allow
the drug–DNA binding free energyto be apportioned in great detail.
One approach involves evaluation of a minimum set of free energy terms, which are assumed to be addi-
tive that can account for the experimentally determined value of Gobs.16,18Based on current concepts of
ligand–DNA interactions, it is reasonable to describe Gobsas being composed of at least five component
free energy terms. Thus,
(9.14)where Gconfis the unfavourable free energy term arising from conformational changes in the DNA and
ligand, Grtis an unfavourable term that results from losses in rotational and translational degrees of free-
dom upon complex formation, Ghydis the free energy for the hydrophobic transfer of ligand from bulk
solventinto the DNA binding site, Gpe(Section 9.3) is the electrostatic (polyelectrolyte) contribution to
the observed free energy that arises from coupled polyelectrolyte effects such as counter-ion release (entrop-
ically favourable) and Gmolis the free energy term arising from weak non-covalent interactions, such as
van der Waals, hydrogen bonding, dipole–dipole interactions, etc.
Each of the terms in Equation 9.14 can be estimated using semi-empirical or theoretical methods. At
least two of the terms, Gconfand Grt, make unfavourable contributions to binding free energy. Kinetic
studies have revealed that the conformational transition in DNA that accompanies binding of a simple inter-
calator such as ethidium has a free energy cost of16.7 kJ mol^1. Therefore, this value can be set as the ener-
getic cost of structurally perturbing DNA to form an intercalation site. When a bimolecular complex is
formed, there is an associated loss of rotational and translational degrees of freedom. This gives rise to a large
entropic cost for binding, which is reflected in the free energy term Grt. There is some debate as to the
precise value for this parameter.^16 However, a value of62.8 kJ mol^1 for Grtfor a rigid body interaction
is not unreasonable. Hence the two unfavourable free energy terms are set as constants for all intercalator bind-
ing reactions.
For a stable ligand–DNA complex to form, the remaining free energy terms must be large enough and
favourable to overcome these two unfavourable contributions. It has been experimentally demonstrated for
intercalators (and groove binders) that the major favourable free energy term that drives binding is the
hydrophobic transfer of drug from the aqueous environment to the DNA binding site. It is possible to quantify
GG GGGGobs conf r t hyd pe mol354 Chapter 9