Discoveries Featured

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 Baskauf.)

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 Baskauf.)

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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.

This debilitating 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.