The Challenge:

Developing therapeutics for CNS disorders, particularly the neurodegenerative disorders, has historically been very difficult.  Compared to cancer, cardiovascular disease or infectious disease, progress in treatment of CNS diseases has been relatively slow, with few new therapeutics being identified.  This lag in innovation in CNS therapeutics can be attributed mainly to the complexity of the brain.  Because the nervous system works through complex circuits involving large numbers of neurons that interact in multiple ways, the molecular and cellular methods that have proved so successful in other disease areas, such as cancer, have not been sufficient for CNS drug discovery and development.

In the past, the search for new therapeutics has also been hampered by lack of understanding of the biological mechanisms for CNS disorders and the lack of good animal models of neurological and psychiatric diseases.  In many cases, cloning of the genes has led to the identification of new proteins and disease pathways, offering new insight into the biological mechanisms of disease.  Most importantly, the elucidation of disease pathways has suggested new potential molecular targets for small molecule pharmaceuticals.

In addition to facilitating mechanistic studies, the identification of human genes for neurological disorders has made the creation of animal models possible, leading to studies on pathogenesis and providing a test system for potential therapeutics.  By expressing a mutated human disease gene in mice, models of Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, ALS, SCA, etc. have been created.  In all cases, the mice exhibit a progressive neurological phenotype characterized by motor dysfunction neurodegeneration and nuclear accumulation and aggregation of the mutant protein.   The use of these mouse models in the drug discovery process however is constrained by costs, space requirements, and time.

A Powerful Solution:

The power and usefulness of Drosophila genetic models of disease has recently been realized (for review see: Zoghbi and Botas, 2002).  By introducing human disease genes with dominant gain-of-function mutations into Drosophila, EnVivo has generated models for a number of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. In each case, expression of the transgene recapitulates many of the essential neuropathological features of the human disease.  For example, in theDrosophila model for Parkinson’s disease, produced by neuronal expression of human mutated alpha-synuclein, age-dependent, progressive degeneration of dopamine-containing cells is seen accompanied by the presence of Lewy bodies, which resemble those seen in the human disease, both by their immunoreactivity for alpha-synuclein and by their appearance under the electron microscope.  In our Drosophila models of Alzheimer’s disease, generated by expressing human beta-amyloid and human tau, the brain shows progressive accumulation of insoluble beta-amyloid, tau hyperphosphorylation, and vesicular aggregation that resemble the human intraneuronal filamentous inclusions. The AD model neuropathology is also accompanied by progressive neuronal loss, learning and memory deficits – all symptoms observed in the human disease state.  In the case of Huntington’s disease, expression of mutant huntingtin fragments, containing an expanded polyglutamine repeat, causes a progressive neurodegeneration, whose time of onset and severity are linked to the length of the repeat, just as is seen in the human disease.

Remarkably, when the human transgenes for neurodegenerative disease are expressed throughout the nervous system of Drosophila, reproducible and scorable behavioral phenotypes results that recapitulates in recognizable ways symptoms of the corresponding human disease.  For example, flies expressing mutated human alpha-synuclein have impaired motor performance; flies expressing the fragment of huntington show uncoordinated, spontaneous movements and hypokinesis.  EnVivo’s Drosophila disease models thus offer specific and well-validated representations of the human diseases.  In particular, the striking recapitulation of behavioral features of the human diseases in flies strongly supports the idea that the functional circuits involved in these diseases have been conserved during evolution.

The tremendous potential for de novo drug discovery through large-scale, in vivo screening has not been exploited until now.  EnVivo has capitalized on this opportunity by developing a high-throughput screening platform for neurodegenerative disease models in Drosophila (Fig. 1).  The strategy of in vivo screening is particularly well-suited to Drosophila because of the extraordinary savings in time and money that they offer and thereby provides a powerful discovery and pre-selection mechanism for subsequent screening in mammals.  EnVivo uses this strategy to carry out large-scale screens, using proprietary genetics and robotics technologies and state-of-the-art pattern recognition software to analyze large numbers of compounds in the Drosophiladisease models.


Schematic of EnVivo’s Proprietary Integrated Discovery Strategy.

Schematic of EnVivo’s Proprietary Integrated Discovery Strategy.

Drosophila is well suited for large-scale screening for the following reasons:

  • Well-validated disease models with symptoms recognizably related to the human disease
  • Large numbers of organisms can be handled reproducibly, efficiently, and cheaply
  • The development of quantitative disease-relevant symptoms over a period of days
  • Rich genetics that can be used to identify targets/pathways affected by active compounds

Several observations support the assumption that active compounds identified in Drosophila are likely to be also active in mammalian systems. First, a comparison of the two genomes suggests that there had been strong conservation of disease genes and pathways during evolution.  Of 929 identified human disease genes, 714 (77%) have closely related genes in theDrosophila genome (Reiter et al., 2001).  Second, a large number of pharmacological agents commonly used by biomedical investigators, including cholinergic and adrenergic agents, protein and RNA synthesis inhibitors, drugs that affect signaling pathways and drugs that affect the cytoskeleton are active in both Drosophila and mammalian systems.  Third, experiments with Parkinson’s disease models in Drosophila have shown that prototypes of the major classes of drugs that are used to treat Parkinson’s disease in humans, including L-DOPA, improve motor function in Drosophila models of Parkinson’s disease (Pendleton et al., 2002).   Fourth, compounds improve disease phenotypes in Drosophila models of Alzheimer’s (Micchelli et al., 2003;  Crowther et. al., 2005) Finally, with respect to trinucleotide repeat disorders, EnVivo and others have demonstrated that compounds against multiple different targets have been shown to be effective in improving function in bothDrosophila and mouse disease models (e.g. Agrawal et al., 2005; Pollitt et al., 2003; Hockly et al., 2003; Ferrante et al., 2003; Minamiyama et al., 2004; Karpuj et al., 2002; McCampbell et al., 2001; Steffan et al., 2001).

In vivo assays impose stringent requirements for bioactivity in that compounds must not only be active, but also bioavailable and non-toxic to the fly.  Thus compounds must be orally available, must pass a barrier, similar to the blood-brain barrier in humans, of the fly to reach the correct site and must not have toxic or debilitating side effects that would obscure performance. The complex requirements for activity, however, are also a major advantage in that the “hits” that are obtained are of very high quality, since they are, by definition, bioactive, bioavailable and safe.   Once a compound is found that is active in the Drosophila models, the compound hit is validated, optimized and tested in vertebrate systems. We believe that the stringent requirements of an in vivo screen means that compounds identified as active in Drosophila will make superior candidates for further development in mammals.   To this end we have developed state of the art capabilities to address the back end of the screening cascades that includes traditional neuropharmacolgy and vertebrate animal model pharmacokinetics and efficacy testing.


EnVivo Pharmaceuticals is leveraging its innovative high-throughput in vivo platform to identify novel small molecule therapeutics.  Our most advanced programs focus on compounds with different mechanisms of actions, such as kinase inhibitors and ion channels.  Several of our programs are expected to be in the clinic in late 2006 and early 2007.