Research Description:
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The research conducted in my laboratory combines both biophysical
and biochemical approaches toward investigating the following four
interrelated programs of research:
- Development of Topoisomerase I-Directed Anticancer Drugs.
The DNA topoisomerases have been established as effective molecular
targets for anticancer drugs. In fact, numerous topoisomerase
II (TOP2)-directed drugs have been in clinical use for many years.
By contrast, only a single family of topoisomerase I (TOP1)-specific
drugs has been introduced into the clinic to date; namely, the camptothecins.
In animal models, the camptothecins have demonstrated a broad spectrum
of antineoplastic activity against solid tumors. Interest
in TOP1 as a new molecular target for anticancer drugs, has stimulated
the search for new TOP1 inhibitors. This search has led to
the identification of numerous new TOP1-inhibiting compounds.
However, despite the increasing number of TOP1 inhibitors, our current
understanding of the molecular mechanism(s) underlying TOP1 inhibition
is still quite limited, a deficiency that hinders our ability to
design new compounds with desired TOP1 inhibiting and tumor cell
killing activities. To address this deficiency, the primary
goal of this research program is to understand the molecular mechanism
by which DNA-binding drugs stimulate human DNA TOP1-Mediated DNA
damage (cleavage).
Relaxation of superhelical DNA by TOP1 involves repeated cycles
of DNA cleavage and religation. One of the most critical steps
in the catalytic cycle of TOP1 is the cleavage step. All known
TOP1 inhibitors have been identified and characterized based on
their ability to trap the cleavage intermediate, the cleavable complex.
This stabilization of the covalent enzyme-DNA cleavage intermediate
is commonly referred to as "TOP1 poisoning." We have found
nitidine, the protoberberines, and the terbenzimidazoles to be the
most attractive among a number of potent TOP1 poisons. Significantly,
these families of ligands are toxic to a number of different cancer
cell lines. We are characterizing the DNA binding and TOP1
poisoning properties of these drugs, while attempting to discern
empirical correlations between specific physiochemical observables
[e.g., binding mode(s), binding energetics, sequence specificity,
etc.] and the expression of TOP1 poisoning and tumor cell killing
activities. However, such information alone is not sufficient
to develop a complete understanding of the complex interactions
that occur within the ternary, (drug-DNA-enzyme) cleavable complex.
Clearly, the most critical information for elucidating the mechanism
of TOP1 poisoning must be derived from characterization of the ternary
cleavable complex. Thus, another of our goals, which we currently
are pursuing, is to determine the molecular forces that govern the
formation and stabilization of the ternary cleavable complex, including
identification of the key drug-DNA, enzyme-DNA, and/or drug-enzyme
interactions that are involved. The information gleaned from
these studies should facilitate the rational design of new drug
analogs with predictably enhanced TOP1 poisoning and tumor cell
killing efficacies.
Click for larger image
X-Ray Crystal Structure of Reconstituted Human
DNA Topoisomerase I in
Covalent Complex with a DNA Duplex
- Characterize the Thermodynamic Driving Forces That Dictate
and Control the Affinities and Specificities of Drugs Designed
to Target Predetermined DNA Sequences.
Small molecules that target specific DNA sequences offer a
potentially general approach for the regulation of gene expression.
DNA-targeted ligands designed for cancer therapeutic applications
must bind a predetermined DNA sequence (e.g., a specific oncogenic
sequence) with high affinity. Polyamides containing the aromatic
amino acids, N-methylimidazole (Im), N-methylpyrrole (Py), and 3-hydroxypyrrole
(Hp), represent a class of small synthetic ligands that can bind
to DNA with affinities and specificities comparable to DNA binding
proteins. Polyamides can be combined in antiparallel side-by-side
dimeric complexes with the minor groove of DNA. The DNA sequence
specificities of these ligands can be controlled by the linear sequence
of Im and Py amino acids. An Im residue on one ligand complemented
by a Py residue on the second ligand recognizes a G•C base pair,
while a Py/Im combination targets a C•G base pair. A Py/Py
combination is degenerate for both A•T and T•A base pairs.
However, a Hp/Py polyamide pair discriminates T•A from A•T base
pairs. Covalently linking polyamide heterodimers and homodimers
within the 2(ligand)-to-1(DNA) motif has yielded designed "hairpin"
ligands with increased affinity and specificity.
Footprinting and affinity cleaving studies have provided information
regarding the orientation and specific affinities of Im-Py polyamides.
Despite the richness of this database, little is known about the
thermodynamic driving forces that dictate the observed binding affinities
and sequence specificities of these ligands. Such knowledge
should prove useful in gene inhibition experiments [where both affinity
(subnanomolar Kd) and specificity (?Kd) are of premium value], since
it provides a basis for the rational design of ligand motifs and
solution conditions that should maximize ligand targeting to the
primary, DNA match site. To this end, our goal is to evaluate
the thermodynamic driving forces that dictate the pairing rules
for polyamide-DNA recognition using hairpin polyamides designed
to target specific DNA sequences ?5 base pairs in length. These
studies represent important first steps toward establishing the
thermodynamic database needed to design polyamides with predictable
DNA binding affinities and sequence specificities.
- Evaluate the Structural, Energetic, and Biological Consequences
of the DNA Lesions Caused by the Anticancer Drug Cisplatin.
cis-Diamminedichloroplatinum(II) (cisplatin) is a widely used
anticancer drug. The chemotherapeutic efficacy of this drug
is derived from its ability to bind and crosslink DNA. The
major DNA adduct of the drug results from coordination of two adjacent
guanine bases to platinum to form the cis-Pt-GG intrastrand crosslink.
Crystallographic, NMR, and electrophoretic studies have revealed
that the cis-Pt-GG crosslink unwinds DNA by 13° and bends it
by 34-55° in the direction of the major groove. Such cis-Pt-GG-induced
alterations in duplex structure have been implicated in the promotion
of specific interactions with cellular proteins that contain one
or more high mobility group (HMG). When bound by such cellular
proteins, the cis-Pt-GG sites are shielded from nucleotide excision
repair, thereby enhancing the cytotoxic efficacy of the drug.
Currently, little is known about the energetic consequences of cisplatin-induced
crosslink formation or about how these consequences are modulated
by the sequence of the bases flanking the crosslink. Such
thermodynamic data should reveal how the crosslink influences DNA
duplex stability, a property that has been implicated in the modulation
of protein recognition and binding. To this end, the goal
of this research program is to characterize the impact of cis-Pt-GG
intrastrand crosslink formation on the conformation and energetics
of duplex DNA, while assessing how this impact is modulated by the
sequence of the bases flanking the lesion.
A logical extension of the studies described above, which
we currently are pursuing, is to characterize the influence of cisplatin
intrastrand crosslinking on the binding of HMG-domain cellular proteins.
Specifically, we are evaluating the thermodynamic driving forces
that govern the binding of HMG1 domain proteins to platinated and
nonplatinated DNA duplexes in which the bases flanking the adduct
are systematically altered. Subsequent correlation of the
resulting thermodynamic data with the structural information about
HMG1-DNA complexes, as derived from NMR and X-ray crystallographic
studies, will provide insight into the energetic and/or structural
basis for protein recognition. The information gleaned from
these studies will facilitate our ability to design novel structure-specific
agents that enhance the cytotoxicity of cisplatin by shielding the
DNA adducts it generates from nucleotide excision repair.
- Define the Molecular Forces that Dictate, Control, and
Stabilize Drug-RNA Interactions.
RNA can fold into a variety of different structures and/or conformations
that can serve as specific recognition elements for drugs.
Targeting these structural RNA elements in a site-specific manner
offers the potential for modulating the biological function of the
targeted RNA. To date, little is known about the thermodynamic
driving forces that dictate, control, and stabilize drug-RNA interactions,
a deficiency that limits our ability to design new agents with predictable
RNA binding affinities and specificities over a range of solution
conditions. The primary goal of this research program is to
define the rules that govern the affinities and specificities of
drugs for their RNA targets. Specifically, we are defining
the relative contributions of van der Waals contacts, hydrogen bonding,
and electrostatic interactions to the binding affinities and specificities
of RNA-directed ligands. We also are evaluating how the presence
and sequence of loops and bulges in the host RNA modulate drug recognition.
Currently, our studies are focused on the aminoglycoside families
of antibiotics. Aminoglycoside antibiotics are bactericidal
drugs used in the treatment of Gram-negative infections. They
inhibit translation by binding to rRNA and blocking the translocation
step of protein synthesis. Significantly, these drugs also
can bind specifically to certain viral RNA molecules (e.g., the
RRE and TAR domains of HIV), highlighting the potential of these
drugs for use as antiviral agents. The information gleaned
from our studies will facilitate the rational design of synthetic
ligands that can modulate the biological functions of RNA molecules
by targeting specific sequences and/or structural motifs.
Click for larger image
NMR-Derived Solution Structure of the Complex
Formed Between the
Aminoglycoside Antibiotic, Paromomycin, and an
A-Site rRNA Model
Oligonucleotide
Recent Publications
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Poklar, N., Pilch, D. S., Lippard, S. J., Redding E. A., Dunham,
S. U. & Breslauer, K. J. (1996) "Influence of Cisplatin Intrastrand
Crosslinking on the Conformation, Thermal Stability, and Energetics
of a 20mer DNA Duplex." Proc. Natl. Acad. Sci. USA 93, 7606-7611.
Pilch, D. S., Poklar, N., Gelfand, C. A., Law, S. M., Breslauer,
K. J., Baird, E. E. & Dervan, P. B. (1996) "Binding of a Hairpin
Polyamide in the Minor Groove of DNA: Sequence-Specific
Enthalpic Discrimination." Proc. Natl. Acad. Sci. USA 93, 8306-8311.
Xu, Z., Pilch, D. S., Srinivasan, A. R., Olson, W. K., Geacintov,
N. E. & Breslauer, K. J. (1997) "Modulation of Nucleic Acid
Structure by Ligand Binding: Induction of a DNA•RNA•DNA Hybrid
Triplex by DAPI Intercalation." Bioorg. Med. Chem. 5, 1137-1147.
Pilch, D. S., Yu, C., Makhey, D., LaVoie, E. J., Srinivasan,
A. R., Olson, W. K., Sauers, R. R., Breslauer, K. J., Geacintov,
N. E. & Liu, L. F. (1997) "Minor Groove-Directed and Intercalative
Ligand-DNA Interactions in the Poisoning of Human DNA Topoisomerase
I by Protoberberine Analogs." Biochemistry 36, 12542-12553.
Sim, S.-P., Gatto, B., Yu, C., Liu, A. A., Li, T.-K., Pilch,
D. S., LaVoie, E. J. & Liu, L. F. (1997) "Differential Poisoning
of Topoisomerases by Menogaril and Nogalamycin Dictated by the
Minor Groove-Binding Nogalose Sugar." Biochemistry 36, 13285-13291.
Pilch, D. S., Xu, Z., Sun, Q., LaVoie, E. J., Liu, L. F. &
Breslauer, K. J. (1997) "A Terbenzimidazole That Preferentially
Binds and Conformationally Alters Structurally Distinct DNA Duplex
Domains: A Potential Mechanism for Topoisomerase I Poisoning."
Proc. Natl. Acad. Sci. USA 94, 13565-13570.
Xu, Z., Li, T.-K., Kim, J. S., LaVoie, E. J., Breslauer, K. J.,
Liu, L. F. & Pilch, D. S. (1998) "DNA Minor Groove Binding-Directed
Poisoning of Human DNA Topoisomerase I by Terbenzimidazoles."
Biochemistry 37, 3558-3566.
Pilch, D. S., Poklar, N., Baird, E. E., Dervan, P. B. & Breslauer,
K. J. (1999) "The Thermodynamics of Polyamide-DNA Recognition:
Hairpin Polyamide Binding in the Minor Groove of Duplex DNA."
Biochemistry 38, 2143-2151..
Plum, G. E., Pilch, D. S., Singleton, S. F. & Breslauer,
K. J. (1995) "Nucleic Acid Hybridization: Triplex Stability
and Energetics." Annu. Rev. Biophys. Biomol. Struct. 24, 319-350.
Pilch, D. S., Plum, G. E. & Breslauer, K. J. (1995) "The
Thermodynamics of DNA Structures That Contain Lesions or Guanine
Tetrads." Curr. Opin. Struct. Biol. 5, 334-342.
Lab Staff
| Kaul, Malvika |
Postdoctoral Fellow |
| Barbieri, Christopher |
Graduate Student |
|
