Download Overview of Current Research

Large Molecule-Small
Molecule Interactions
DNA
Proteins
Targeting DNA or RNA
i) Alter regulation of replication, transcription or translation
ii) Kill cells, not alter regulation
General problems
i) Poor tissue specificity, leading to side effects and high
toxicity
ii) Mutagenic, teratogenic and carcinogenic properties
Important
aspects of DNA
and RNA
structure
Watson-Crick base pairs
Minor and Major Grooves
Phosphodiester backbone
Conformational flexibility
DNA intercalators
DNA minor groove binders
Structure of Distamycin A bound to DNA
Combine intercalation with minor groove
binding
Toxicogenomics: Overview and potential
applications for the study of non-covalent
DNA interacting chemicals
Heng-Hong Li, Jiri Aubrecht, Albert J.
Fornace Jr.
Department of Biochemistry and Molecular and Cellular Biology and the Lombardi
Comprehensive Cancer Center, Georgetown University, Washington, DC 20007, USA
Pfizer Global Research and Development, Groton, CT 06340, USA.
What is Toxicogenomics?
•
Toxicogenomics is a form of analysis by which the activity of a particular toxin or chemical
substance on living tissue can be identified based upon a profiling of its known effects on
genetic material. Once viable, the technique should serve for toxicology and toxin-determination
a role analogous to DNA-testing in the forensic identification of individuals.
•
A field that utilizes gene responses to define expression profiling signatures for various types of
drugs and toxicants, and to provide mechanistic insight into their cellular effects.
•
Basically, toxicogenomics measures some sort of cellular activity in response to a drug in order
to determine what effect that drug has on the DNA.
•
THIS PROJECT: measures some sort of response (melting shift, electrophoresis
mobility) of DNA to a small molecule (e.g. a drug) that may indicate cellular activity of
that small molecule.
Why?
CANCER RESEARCH!!
Covalent chemical interactions
Non-Covalent interactions
Direct damage to DNA by
forming adducts or strand breaks.
Perturb DNA and chromatin.
BOTH
Lead to genetic changes that can contribute to cancer
development.
New Testing Techniques
Needed
Former Technique:
Problems Today:
Low specificity for predicting
carcinogenicity in vitro mutation and
chromosome damage assays.
Genotoxicity testing battery
Since 1970’s effectively assured
genetic safety of consumer
chemicals and drugs.
Sensitive, simple, fast and
economical.
False Positives
Positive drug genotoxicity results
and negative carcinogenicity results
tested positive for in vitro and
damage assays.
SO
The interpretation of these findings
presents a major challenge to
industry and regulatory agencies.
Why new techniques?
Threshold Response
Agents that do not directly interact with the DNA exhibit a threshold dose response
and should not be assessed based on linear extrapolation approaches that are used
for agents that directly interact with the DNA.
Differentiating a true threshold dose from reaching an
assay detection limit.
Need an understanding of the underlying genotoxic mechanisms. This is a challenge
because it takes a lot of time, work, and the outcomes are uncertain. The results
are delays in drug developments.
Therefore, new techniques for the advancement of alternative experimental
approaches capable of evaluating a whole range of genotoxic mechanisms is
important.
Structure = Function!
(Basic Organic still reigns supreme)
Covalent
Non-Covalent
Irreversible interaction
Reversible interaction
Unrepaired base damage:
Van der waals, hydrogen bonding,
hydrophobic, charge transfer forces.
Mutagenic and lethal consequences, such as mismatch
repair or production of apurinic/apyrimidinic sites.
DNA backbone distortion affecting transcription and
replication
Groove binders:
Crescent shaped, not toxic.
Intercalators:
Planar aromatic, DNA backbone distortion,
frameshift mutations during replication, change of
torsional strain in cellular DNA.
Non-Covalent Complications
•
•
•
•
The inclusion of other damaging properties with NC chemicals.
NC chemicals can be just as, or more, toxic than covalent chemicals.
NC chemical interactions with DNA are complex.
Broad mechanisms-based scientific approaches, such as toxicogenomics
methodology, will be advantageous for providing insights into the multifaceted nature of toxic mechanisms of NC DNA interacting agents.
Groove Binding Agents
Berenil
Chloroquine
Chromomycin A3
Distamycin A
Hoechst 33258
Bleomycin
DAPI (Diamidine-2-phenylindole
Pentamidine
Netropsin
Aminoacridine
DNA Intercalators
Napthalimide
Ethidium Bromide
Coumarin
Indole
Phenanthridine
Doxorubicin
M-AMSA
Proflavine
Daunomycin
Quinoline
Mitoxantrone
Quinoxaline
Covalent DNA Interaction Molecules
Busulfan
Nitrogen Mustard
Camptothecin
Nitrosourea
Chlorambucil
CCNU
Cis-platinum
PCNU
Previous Categorization of Mechanisms of
Action
Categorize molecules by their biochemical mechanisms of
actions.
Six general mechanisms: Alkylating agents, topisomerase 1 inhibitors,
topoisomerase 2 inhibitors, RNA/DNA antimetabolites, DNA antimetabolites,
antimitotic agents.
Yeast studied.
Damage by simple alkylating agent.
Approach used to assign unknown agents to these 6 categories
and other categories.
New Categorization of Mechanisms of
Action
Genotoxic stress responses now being used in
research, particularly transcriptional stress
responses.
p53 transcriptional response is a common
stress response measurement.
p53, also known as protein 53 (TP53), is a transcription factor that regulates the cell
cycle and hence functions as a tumor suppressor. It is important in multicellular
organisms as it helps to suppress cancer. p53 has been described as "the guardian
of the genome", "the guardian angel gene", or the "master watchman", referring to its
role in conserving stability by preventing genome mutation.
Intercalators and Groove Binders:
How they affect transcriptional responses
• The capability of disrupting interaction between transcription
factors and DNA varies among the non-covalent agents
depending on the compound structure, side chain, sequence
preference, and affinity to DNA.
• Intercalating agents, such as Ethidium bromide, can also affect
mitochondrial DNA and function.
• Previous studies imply that recruitment of transcriptional factors
to promoters may be affected by DNA torsional strain from the
effect of non-covalent agents.
Future Research
• Studies on non-covalent interacting agents need to be run. The
results for current and past studies are limited.
• Non-covalent interacting chemicals have relatively low cytotoxicity
and consideration must be taken when interpreting results where a
particular agent may have additional mechanisms of action.
• Must evaluate more subtle effects from NC agents for changes in
DNA torsional tension and chromatin structure.
• Reliable results in large data sets will be needed to define signatures
for different types of NC DNA interacting agents.
DNA Melting Simulations by
Molecular Dynamics
(AT)n dsDNA + APS
10ps to 300K
DNA Tm Obs vs Tm MD
410
400
y = 1.4222x + 293.02
2
R = 0.8752
Tm MD
390
380
T(K)
Linear (T(K))
370
360
350
340
35
40
45
50
55
Tm Obs
60
65
70
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
The Other Big Molecules:
Proteins
Acetylcholinesterase
[The following slides are of AchE
taken from the Protein Data Bank.
The ligand is a competitive
inhibitor]
EVIDENCE OF INHIBITION
pH
AchE Assay 10/24/01
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
No Enzyme
y = -0.008x +
8.1731
R2 = 0.9815
Enzyme Added
y = -0.0116x +
9.5723
Series1
[ I ] Added (p-Xylyl)
y = -0.0014x +
5.587
0
200
400
600
Time(sec)
800
1000