complex is that it requires the minor groove to widen two-fold compared to drug-free DNA or to the 1:1 com-
plex (Figure 9.14). This structural rearrangement gives rise to a positive free energy contribution to bind-
ing, in a similar way to structural deformations that accompany intercalation. However, this unfavourable
free energy is more than compensated by additional favourable free energy that arises from the greater
hydrophobic interactions derived from binding of two drugs within one minor groove. This shows another
example of the conformational mobility of DNAthat is crucial to the interaction of proteins and ultimately
to biological function.
9.7.2.2 Energetics of Netropsin– and Distamycin–DNA Interactions. Recently, there has been an
upsurge in detailed thermodynamic studies of minor groove binding that go beyond simple determinations of
binding constants. For netropsin, distamycin, and a number of other minor-groove specific ligands, it is now
possible to apportion the overall binding free energy intoenthalpic and entropic terms. In addition, recent
developments in thermodynamic analysis allows a much more detailed parsing of free energy terms (Section
9.6.4 and Equation 9.14), which provides insight into the energetics of drug–DNA interactions.
The relative thermodynamic contributions of two distamycin molecules that bind to the duplex
d(GCCAAATTGGC) d(GCCAATTTGGC), referred to as A3T2, have been examined in the context of the
thermodynamics of the 1:1 netropsin interaction with A3T2.^34 In all cases, drug binding is associated with an
exothermic binding enthalpy, which for netropsin is 31.4 kJ mol^1 and for distamycin first and second bind-
ing events are 51.5 and 78.7 kJ mol^1 , respectively. The drugs bind to DNA moderately tightly with Kobs
of 4.3 107 M^1 for netropsin and 3.1 107 M^1 for the first distamycin binding. The second distamycin
molecule binds with a lower affinity of 3.3 106 M^1 (20°C, pH 7.0 and Naconcentration of about 20 mM).
Thus netropsin and the first distamycin have very similar overall binding affinities and both interactions
are enthalpically driven, with the first distamycin having a larger favourable enthalpy. One possible
interpretation for these observations is that hydrogen bonding contributions are similar for the two com-
plexes. The smaller number of favourable electrostatic interactions in the monocationic distamycin, com-
pared to dicationic netropsin, is compensated by an increase in hydrophobic interactions in distamycin
arising from its additional N-methylpyrrole ring. The magnitudes of the negative enthalpy values are
dependent upon relative contributions from hydrogen bonding, van der Waals interactions, and overall
hydration changes. The entropy contributions are of opposite sign (favourable for netropsin and
unfavourable for distamycin), probably due to differences in release of counter-ions and uptake or release
of water molecules. The second distamycin (in the 2:1 complex) binds with an eight-fold reduction in
affinity and a 27.2 kJ mol^1 more favourable enthalpy. This finding is consistent with observations from
the NMR structure, since there is an increase in van der Waals contacts due to side-by-side interactions in
the 2:1 distamycin–DNA complex.
Variations in binding affinity with salt concentration are also revealing. The slopes of lnKobsversus
ln[Na] plots were found to be 0.8 for netropsin–A3T2 binding and 0.56 for distamycin–A3T3 bind-
ing. These values are lower than previous estimates for 1:1 binding of netropsin and distamycin to A2T2
and also lower than the theoretical value of 1.76 (for the binding of one dicationic ligand or two mono-
cationic ligands) predicted from the polyelectrolyte theories of Record and Manning. Structural data pro-
vides a possible explanation for these results. The minor groove becomes widened upon the binding of the
second distamycin, which results in a lower local charge density. Also, structured water around exposed
hydrophobic groups of the bound drug may give rise to a different type of dielectric screening. For both
ligands, calorimetrically measured binding-induced changes in heat capacity Cpare significant in mag-
nitude and negative in sign, consistent with theoretical estimates based on changes in solvent accessible
surface area upon binding.^35
9.7.3 Lexitropsins
Lexitropsinsare a group of minor groove-specific ligands that are comprised of a range of different aromatic
rings connected by peptide bonds. Netropsin and distamycin are examples of lexitropsins, where the sequence
364 Chapter 9