Vonetta Edwards, Kathy Goodson, Habi Mojidi and Sarah Mae Sucayan Biochemistry 674, University of Maryland, College Park, MD

1
Designing Minor Groove Binding Drugs
Vonetta Edwards, Kathy Goodson, Habi Mojidi and
Sarah Mae Sucayan Biochemistry 674, University
of Maryland, College Park, MD
Current Strategies
Introduction
Factors in Recognizing the Minor Groove
Electrostatics

• Transcription or replication of DNA only
  occurs after a signal has been received, usually
  in the form of a protein that binds to a
  particular region of the DNA. If a small
  artificial protein can be developed that would
  mimic the binding strength and specificity of the
  natural regulatory protein, then DNA function can
  be artificially modulated, inhibited or
  activated. Drugs may bind irreversibly, or
  reversibly in which non-covalent bonds are
  formed. The latter is preferred when developing
  drugs.1-3
• Minor groove binders are crescent shaped, bind
  via Van der Waals interactions and hydrogen
• bonds and have a preference for AT rich
  sequences. e.g. distamycin, Hoescht 33258.
• Intercalators consist of planar
  heterocyclic/chromopore groups that stack between
  adjacent DNA bases and also have hydrogen
  interactions. Most prefer GC rich regions
  (bleomycin).

• A/T sequences (Figure 2A) have a higher
  negative
• potential while G/C sequences (Figure 2B)
  have
• higher positive potential
• H-bonding to base edges in the minor groove
  can
• be used to distinguish between AT and GC
  base
• pairs
• AT is distinguished from a TA base pair
  through
• indirect read out

One of the first chemical approaches to targeting
double-stranded DNA used oligonucleotides linked
to intercalators to bind in the major groove of
DNA and form a sequence specific triple helix.4
A
B
Figure 74 Triple Helix Model
Categories of Sequence Specific DNA Binding Drugs
Figure 25 Electrostatic potential surface of
dsDNA crystal structures.
Another strategy in improving sequence
specificity of minor groove binding drugs is the
use of polyamides. These group of molecules
contain combinations of three different aromatic
amino acids (hydroxypyrrole, imidazole, pyrrole),
which uniquely bind with each of the Watson-Crick
base pairs.(see Figure 9).4
Structure

• Triple Helix forming molecules
• Synthetic Polyamides
• Zn Finger proteins

Width Depth
Major Groove 11.6Å 8.5Å
Minor Groove 6.0Å 8.2Å
Figure 84 Hairpin Polyamide Model
Figure 14Interaction of the different
combinations of amides with specific WC base
pairs.
Figure 3 Left Groove dimensions5. Right WC
base pairs.7
Biological Effects of DNA Binding Drugs

• DNA binding drugs are generally flat and small,
  which makes them fit well into the
• minor groove.
• Groove width varies with sequence A/T rich
  tracks tend to make the groove width
• narrower.
• The convex shape of the minor groove floor
  complements the typical shapes of minor
• groove binding drugs
• A/T sequences result in a smooth convex curve
  whereas G/C sequences have little
• bumps due to the 2-amino groups of guanine.

Transcription factors The most effective
inhibitors bind to both the major and minor
grooves and allow threading which keeps the DNA
inaccessible to transcriptions factors.1 Topoisom
erases cleavable complex poisons ,such as
quinolones, stabilize covalent enzyme-DNA
complexes thus prevent resealing of DNA ends.
This converts type II enzymes into a cellular
poison..catalytic inhibitors, such as
coumermycin, inhibit essential ATP hydrolysis
needed for topoisomerase function.3
Figure 9 Molecules such as Pyrole-imidazole
polyamides are linear Beta-alanine linked
polyamides that recognize a large range of DNA.12

Initial Probe Studies
Conclusion and Future Work
Beginning with the work of Galas and
Schmitz, methods of DNA footprinting1 with DNase
I allowed for determination of the location of
molecular binding sites on DNA.8 A synthetic
cleavage agent methidiumpropyl-EDTA (MPE) is
comprised of methidium, a DNA intercalator,
which is covalently bound to the metal chelator
EDTA. This agent was later adopted for use in
footprinting due to its smaller size and its
preferable mode of activation.9
DNA binding molecules have various
affinities for specific regions of DNA.
Synthetic analogs of the AT-selective minor
groove-binding ligands13 created the foundation
for synthetic DNA binding drugs. Sequence
specificity of DNA binding drugs will provide
insight into drug design that will target genes
and be used as a class of potential therapeutics
against unknown biological weapons and
personalized medicines.14 Currently, various
clinical trials of genetic therapies are in
progress to find effective, safe designer drugs
to target various harmful genetic conditions.
Once some of the conditions have been met this
will lead to the possible eradication of some
genetic disorders.
References
Figure 411 MPE
The mode of cleavage of EDTAFe(II) is
through the use of a non-specific hydroxyl
radical, thus cleavage is attributed to the
binding ligand. In experiments by Younguist and
Dervan, EDTA was attached to either the amino or
carboxyl end of tri-, tetra-, penta-, and
hexa(tris-N-methylpyrrolecarboxamide)s. The
result of experimentation was the outline of an
n1 binding motif, where n is the number of
amides.10
1. Bassi, L. Palitti, F., Anti-topoisomerase
drugs as potent inducers of chromosomal
aberrations. Genetics and Molecular Biology 2000,
23, (4), 1065-1069. 2. Gambari, R. Feriotto,
G. Rutigliano, C. Bianchi, N. Mischiati, C.,
Biospecific interaction analysis (BIA) of
low-molecular weight DNA-binding drugs.
Journal of Pharmacology and Experimental
Therapeutics 2000, 294, (1), 370-377. 3. Welch,
J. J. Rauscher, F. J. Beerman, T. A., Targeting
DNA-Binding Drugs to Sequence-Specific
Transcription Factor DNA Complexes
Differential-Effects of Intercalating and
Minor-Groove Binding-Drugs. Journal of Biological
Chemistry 1994, 269, (49), 31051-31058. 4. Uil,
T. G. Haisma, H. J. Rots, M. G., Therapeutic
modulation of endogenous gene function by agents
with designed DNA-sequence specificities.
Nucl. Acids Res. 2003, 31, (21), 6064-6078. 5.
Neidle, S., DNA minor-groove recognition by small
molecules. Natural Product Reports 2001, 18, (3),
291-309. 6. Moser, H. E. Dervan, P. B.,
Sequence-specific cleavage of double helical DNA
by triple helix formation. Science 1987, 238,
(4827), 645-650. 7. Dickerson, R. E., In Oxford
Handbook of Nucleic Acid Structure, Neidle, S.,
Ed. Oxford University Press 1999 pp 145-197. 8.
Galas, D. J. Schmitz, A., DNAse footprinting a
simple method for the detection of protein-DNA
binding specificity. Nucleic Acids Res 1978, 5,
(9), 3157-70. 9. Van Dyke, M. W. Dervan,
P. B., Methidiumpropyl-EDTA.Fe(II) and DNase I
footprinting report different small molecule
binding site sizes on DNA. Nucleic Acids Res
1983, 11, (16), 5555-67. 10. Youngquist, R. S.
Dervan, P. B., Sequence-Specific Recognition of
B-DNA by Oligo(N-methylpyrrolecarboxamide)s. PNAS
1985, 82, (9), 2565- 2569. 11. Dervan, P.
B., Design of sequence-specific DNA-binding
molecules. Science 1986, 232, (4749), 464-71. 12.
Burnett, R. Melander, C. Puckett, J. W. Son,
L. S. Wells, R. D. Dervan, P. B. Gottesfeld,
J. M., DNA sequence-specific polyamides
alleviate transcription inhibition
associated with long GAAmiddle dotTTC repeats
in Friedreich's ataxia. PNAS 2006, 103, (31),
11497-11502. 13. Schaal, T. D. Mallet, W. G.
McMinn, D. L. Nguyen, N. V. Sopko, M. M. John,
S. Parekh, B. S., Inhibition of human papilloma
virus E2 DNA binding protein by covalently
linked polyamides. Nucleic Acids Research 2003,
31, (4), 1282-1291.
Figure 6EDTAFe(II) affinity cleavage model
reflecting the asymmetric DNA cleavage pattern
seen in the minor groove of B-DNA. The frequency
of cleavage is represented by the lengths of the
arrows. Figure adapted from Youngquist and
Dervan.10
Figure 511 Amino and carboxyl labeled
tris-N-methylpyrrolecarboxamide