Drug Discovery Focuses on Fish
By: Carol A. Rouzer, VICB Communications
Published: February 5, 2010
An innovative approach to drug discovery capitalizes on the developing zebrafish embryo and offers hope to sufferers of bone diseases and cancer.
The discovery and development of new drugs is a complex, challenging, and expensive process. Since most drugs work by altering the function of a target protein (usually an enzyme or receptor), the first task is to find a molecule that has the desired activity. Then one must be certain that the molecule does not
influence the function of any other proteins, that it can be
delivered to the appropriate tissues, that it is not too rapidly
metabolized, and that it is not toxic. Because each of these
goals is addressed individually during the development process,
a promising new molecule that has the desired activity at the
protein target is often later found to be unsuitable due to side
effects, excessive metabolism, poor absorption, or toxicity. Now
Vanderbilt Institute of Chemical Biology members Charles Hong and Craig Lindsley report on a
drug discovery innovation that integrates assessments of activity, specificity, absorption, and
toxicity into a single screen.
Figure 1. Adult zebrafish (Dania
rerio). (IImage courtesy of Vanderbilt
Bioimage . Copyright 2002 Steve
Key to the new approach was the realization that the
developing zebrafish (Figure 1) provides an excellent model
system in which to observe the activity of potential new drugs.
Zebrafish produce huge numbers of eggs, which upon
fertilization, develop into embryos that are visible through a
transparent membrane (Figures 2 and 3). Normal embryonic
development is a carefully orchestrated process involving the
interaction of multiple complex signaling pathways. Alteration of
the function of a key protein in any single pathway leads to
abnormalities, which can often be assessed in zebrafish embryos by simple microscopic observation. Because zebrafish
are easy to manipulate genetically, gene knockout or
overexpression techniques can be used to change the levels of a
target protein so that its impact on the development process can
be assessed. Then potential drug candidates can be screened
for their ability to elicit the same effect. Drugs are administered
by simply adding them to the water, so this method also
measures a potential drug’s solubility and capability to
penetrate cellular membranes. Toxicity and side effects are
revealed through the development of unexpected abnormalitiesor generalized developmental retardation.
PUFAs are characterized by a long chain of carbon atoms, and the presence of two or more carbon-to-carbon double bonds. The double bonds render PUFAs susceptible to ROS attack, which breaks the carbon chains into fragments that are themselves highly reactive and capable of inflicting further damage. Some of these fragments stay within the cell membrane where they damage other lipids or membrane proteins. Other fragments are free to move throughout the cell, including into the nucleus where they can damage DNA (Figure 2).
Figure 2. Zebrafish embryo at
about 4 h. Figure 3. Zebrafish
embryo at about 48 h.
(Image courtesy of Vanderbilt
Bioimages. Copyright 2002 Steve
Baskauf.) (Image courtesy of Vanderbilt
Bioimages. Copyright 2002 Steve
To test the value of this approach Hong and co-workers
focused on the effects of bone morphogenic proteins (BMPs),
which promote bone growth, and also play a role in multiple
aspects of embryonic development. BMPs act by binding to
specific receptor proteins. Genetic studies showed that blocking
the activity of these receptors in zebrafish led to a developmental
abnormality called dorsalization in which the dorsal (top) side of
the embryo is overdeveloped relative to the ventral (bottom) side.
Initial screens for compounds that caused embryo dorsalization
led to the discovery of dorsomorphin (Figure 4), which proved to
be an inhibitor of BMP receptors [P.B. Yu et al. (2007) Nat. Chem.
Biol., 4, 33]. However, further studies demonstrated that
dorsomorphin also interfered with the development of blood
vessels in zebrafish embryos. This led the investigators to
question whether BMP signaling is required for normal blood
vessel development in the zebrafish.
To answer this critical question, the Hong lab exploited the
observation that adding dorsomorphin to embryos 4 h after
fertilization resulted in dorsalization whereas adding it 12 h after
fertilization led to vascular abnormalities. Thus by varying the
time of addition, they could differentiate the two effects of the
compound. This provided a way to screen libraries of new
molecules synthesized by the Lindsley lab in a search for
compounds that would exert only one of the two effects. Their approach proved successful and
produced new, more potent, and less toxic inhibitors of BMP, as well as selective inhibitors of
blood vessel development (e.g. DMH1 and DMH4, respectively, Figure 4) [J. Hao et al. (2009) ACS Chem. Biol., published online Dec. 20, DOI: 10.1021/cb9002865].
Figure 4. Structures of
dorsomorphin, DMH1 and DMH4.
These results illustrate the advantages of the zebrafish embryo screen as a means to
evaluate efficacy, specificity, and toxicity of compounds in a single system. By revealing the
ability of dorsomorphin to cause dorsalization and inhibit blood vessel formation, the screen
impelled the collaborators to develop new compounds that affect each pathway individually.
Consequently, the investigators are on the path to drugs that offer hope for sufferers of diseases
of abnormal bone metabolism such as fibrodysplasia ossificans progressiva.
inherited disease is characterized by bone formation at sites of tissue repair after injury so that,
over time, a patient’s body appears to “turn to stone”. The discovery that the disease is caused
by overactivity in the BMP signaling pathway suggests that inhibitors such as DMH1 may offer
relief to these patients. Equally exciting is the realization that selective inhibitors of vascular
development such as DMH4 may be valuable in the chemotherapy of cancer.