Vol. 1, No. 1 2004
Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Target identification
Model organisms and target discovery Marco Muda*, Sean McKenna Serono Reproductive Biology Institute Inc., One Technology Place, Rockland MA 02370, USA
The wealth of information harvested from full genomic sequencing projects has not generated a parallel increase in the number of novel targets for therapeutic intervention. Several pharmaceutical companies have realized that novel drug targets can be identified and validated using simple model organisms. After decades of service in basic research laboratories, yeasts, worms, flies, fishes, and mice are now the cornerstones of modern drug discovery programs.
Introduction To answer fundamental questions about complex biological processes, biologists have tried to identify and develop simple and tractable model systems. Breakthrough discoveries made by studying prokaryotes paved the way to the studies of more complex organisms like yeasts, nematodes, flies, fishes, mice, and humans. Somewhat paradoxically, little use has been made of these same organisms in the development of drugs. With new light shed from full genome sequencing projects, the use of model organism in drug discovery is getting a new boost. Indeed, one of the main outcomes of the genomic sequencing projects is the recognition that many human genes, including those associated with diseases, are conserved in evolution from yeast to man. More specifically, full genomic sequence comparisons have in fact revealed that 46% of Saccharomyces cerevisiae, 43% of Caenorhabditis elegans, 61% of Drosophila melanogaster, 80% of zebrafish, and up to 97% mouse genes are similar to human genes [1]. This should not be astonishing because biological research had already demonstrated that many of the mechanisms developed by prokaryotic and eukaryotic cells to use energy, regulate gene *Corresponding author. (M. Muda) [email protected] 1740-6749/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2004.08.001
Section Editors: Wolfgang Fischer, Rob Hooft, Michael Walker For ethical and practical reasons, we use model organisms in the search for drugs that are, in the end, to be used in humans. Which model organism to choose is a trade-off between similarity to ourselves (chimps, other primates), versus ease of use and manipulation (Escherichia coli). Between these two extreme examples are choices that prioritize being a simple, fast-breeding eukaryote (yeast), insect (body plan/flies), vertebrate development (nematode or zebrafish) or small mammal (mouse). It is important to realize that the basis for these choices evolves with our understanding of biology and disease; for instance, bacteria were studied in the 1960s and 1970s to understand replication, transcription, and translation, but the trend has since moved towards more complex models. This review presents an overview of the major model organisms that are in vogue today, with an emphasis on how they are used to discover conserved signaling mechanisms.
expression and respond to environmental challenges utilize similar basic biochemical processes. Nature has invented a limited number of biochemical mechanisms regulating cellular processes and these same mechanisms have been used, reused, and modified during evolution. The concept of biochemical conservation is not novel and, in fact, the French Nobel laureate Jacques Monod remarked in 1965, ‘‘What is true for E. coli is true for an elephant.’’ This notion has permeated and was the foundation of molecular biology research that began in the first half of the previous century. The use of simple model organisms to dissect complex biological processes has permitted biology to advance at an impressive pace, and the knowledge generated by integrating genetic and biochemical studies has allowed scientists to begin to understand the molecular basis of complex diseases such as cancer and diabetes. Several pharmaceutical companies have started developing research www.drugdiscoverytoday.com
55
Drug Discovery Today: Technologies | Target identification
Vol. 1, No. 1 2004
Table 1. Model organism in drug discovery comparison Common name (scientific name)
Baker’s yeast (S. cerevisiae)
Round worm (C. elegans)
Fruit fly (D. melanogaster)
Zebrafish (Danio Rerio)
Mouse (Mus musculus)
Genome size (genes number)
6000
20,000
14,000
In progress
35,000
Whole genome sequence
Yes
Yes
Yes
In progress
Yes
Generation time
2h
3 days
10 days
3 months
3 months
HTS whole animal assay 96 well format
Yes
Yes
Yes, larva stage
Yes, larva stage
No
Gene silencing
Homologus recombination Transposon
RNAi
Homologus recombination Transposon RNAi
Morpholino
Homologus recombination
Homologous recombination = reaction between two DNA molecules used to modify or disrupt the sequence of a gene at its specific location in the genome. Transposon = mobile genetic element used to disrupt or alter gene expressions.
programs that use simple organisms to identify and validate drug targets. There are five very popular model organisms that can be used to discover novel drug targets, and each has a distinct collection of molecular tools (see Table 1 below). The complete genomic sequence is known for most of them, and using specific molecular genetic methods a genomic wide screen can be designed to identify novel genes relevant for human diseases. Because of their small size, short generation time, high fecundity and cost effective maintenance, certain model organisms can also be used for high-throughput screens (HTS) of small molecules. In fact, in many cases, standard microtiter plates (96 wells) can be used for whole animal assays to assess toxicity, identify novel therapeutic compounds, and unravel drug mechanisms of action. This review will focus on the use of model organisms, giving some examples demonstrating the use of model systems for the dissection of biological processes relevant for human diseases. Furthermore, when possible, it will illustrate some recent reports revealing how model organisms can be used to advance drug discovery.
Baker’s yeast as a model organism S. cerevisiae (Baker’s yeast) has been extensively used to study the molecular basis of cell division and gene regulation in eukaryotic cells. S. cerevisiae has been instrumental in the development of technologies that allow the identification and dissection of protein–protein interactions using technologies such as two-hybrid screens. Moreover, the study of yeast biology has also been instrumental in understanding signaling pathways such as G-protein coupled receptors and protein kinases, both of which are important targets in drug development. One kinase-based signaling cascade that in recent years has benefited from basic discoveries in yeast is the mitogenactivated protein kinase extracellular or receptor-stimulated 56
www.drugdiscoverytoday.com
kinase, MAPK/ERK. The first member of this family was isolated from insulin-treated adypocytes [2]. MAPKs are activated by MAPK kinases (MAPKK) which themselves are dependent upon a further upstream kinase, MAPKKK. The first kinase responsible for MAPK activation was earlier identified as a mutant in the mating response of S. cerevisiae STE7 [3]. The MAPK cassette, composed of three kinases sequentially activated, is now recognized as a universal signaling module vital for cellular adaptation, proliferation and differentiation. Elements of the MAPK pathways are now recognized targets for drug development for treating cancer, inflammation and immune diseases. Not only has S. cerevisiae been important for the early fundamental discoveries in the MAPK cascade; recently, scientists at Pfizer have used S. cerevisiae to clarify the mechanism of action of PD 184353, a small-molecule inhibitor of MAPK/ERK kinase, MEK, the human MAPKK [4]. MEK inhibitors are promising novel compounds that are in development for the treatment of several types of tumors and have a demonstrated ability to block growth of colon cancer in mice [5]. To identify the domain recognized by the inhibitor PD 184353, Delaney et al. reconstituted the human MAPK signaling cascade in yeast, conferring the ability to grow in the absence of one amino acid. A library of mutated MEK was generated and screened for mutations that conferred resistance to the inhibitor. Several mutants were identified and this information, together with structural modeling, identified a novel hydrophobic pocket that is likely to be involved in the binding of PD 184353.
A worm as a model organism In the early 1960s, after breakthrough discoveries in molecular biology had solved fundamental questions in biology, Sydney Brenner proposed to use the nematode C. elegans to begin attacking the problems of cellular development. C. elegans was chosen because the small, transparent nematode
Vol. 1, No. 1 2004
was amenable to microscopic visualization to follow the development and fate of all the 1090 cells that are generated during early development. During C. elegans development, 109 cells are lost by programmed cell death or apoptosis. This characteristic made C. elegans a useful genetic model for the identification of the genes involved in the regulation of programmed cell death. In cells destined to die, the level of the egg-laying abnormal-1 protein, EGL-1, is increased. EGL-1 interacts with a protein complex composed of cell death abnormal-9, CED-9 (similar to human B-cell lymphoma-2, BCL-2) and CED-4, releasing CED-4 (similar to human apoptotic protease activating factor-1, Apaf-1) that in turn activates CED-3 (similar to human Caspases). As in humans, C. elegans programmed cell death is a major feature of the normal development of the nervous system. Importantly, uncontrolled cell death is thought to be the cause of several neurological disorders such as stroke and neurodegenerative diseases. Underscoring the importance of groundbreaking work in C. elegans on the molecular basis of development and apoptosis, Sydney Brenner, Robert H. Horvitz, and John E. Sulston were awarded the Nobel Prize in Physiology or Medicine in 2002. In addition to its fundamental contribution illuminating the process of programmed cell death, C. elegans has been instrumental in the discovery of double-stranded RNA interference (RNAi), which has now become a widely used method in gene functional studies from fly to human. Discovery of this phenomenon came from antisense RNA experiments, where it was observed that both sense and antisense RNAs were effective at producing pheno-copies of genetic loss-offunction mutations. It was later determined that combination of sense and antisense strands, producing dsRNA, were necessary for potent and specific knock-down of protein expression [6]. C. elegans has just started to be explored for drug development, but it has already shown potential in the identification of novel drug mechanisms of action. A genetic screen based on C. elegans has demonstrated that Prozac might have additional in vivo effects distinct from the well-accepted role in blocking serotonin re-uptake. It remains to be seen whether other anti-depressants that block serotonin reuptake have similar unexpected activities that might have implications in the clinical side-effect profiles of this class of drug [7].
The fruit fly as a model organism Due to ease of culturing and its high fecundity, D. melanogaster has been the workhorse of geneticists since T.H. Morgan’s seminal work at the beginning of 1900. Many of the foundations of modern biology have been inspired by the discoveries made with this small insect. Indeed, in 1995 Christiane Nusslein-Volhard and Eric Wiechaus were awarded the Nobel Prize for their groundbreaking research
Drug Discovery Today: Technologies | Target identification
aimed at the identification of the genes involved in embryonic development. In recent years, several original discoveries made during studies in Drosophila have shed light on complex human diseases such as cancer and diabetes. In fact, study of the oncogene ras (identified from rats with sarcoma) in this model has identified novel genes that modulate the same signaling pathway in man. Mutant forms of ras were among the first oncogenes identified; three distinct ras genes named H-ras, K-ras and N-ras are encoded in the human genome. Ras proteins belong to a large family of small GTP-binding proteins that regulate diverse cellular functions. Normal Ras proteins can bind either GDP or GTP, and mutations of Ras that lock the protein conformation in the GTP-bound state are frequently found in human cancer. Ras has been shown to associate and activate Ras activated factor, Raf, a MAPKKK upstream of MEK (see above), and constitutive activation of this pathway is sufficient to drive cell transformation and tumor formation. Importantly, the guanine nucleotide exchange factor responsible for the exchange of GDP to GTP on Ras, named Son-of-Sevenless (Sos), was identified in a Drosophila genetic screen aimed at identifying genes involved in the differentiation of photoreceptor cells. As in mammalian systems, Sos acts downstream of receptor tyrosine kinases, whereas sevenless is the gene encoding the receptor essential for photoreceptor differentiation. Drosophila has the additional advantage that cell lines have been established that can be used to perform biochemical experiments that complement genetic studies. Remarkably, RNAi has been extensively implemented using Drosophila cell lines and this powerful yet simple technology has been used to dissect signaling pathways and to identify novel genes regulating cytoskeleton dynamics [8,9].
A fish as a model organism Because of its relatively small and compact genome size, Fugu rubripes, commonly known as puffer fish, made an attractive model for comparative genomic studies [10]. However, because of its relative large size and slow reproductive rate, Fugu has not yet been widely used as a tool in drug discovery. By contrast, Danio rerio (zebrafish) has many features that render it an attractive alternative to other model organisms, including high fecundity, extra uterine development and transparency of the embryo allowing direct observation of internal organs during early stages of development. Zebrafish larvae can live in less than 100 ml of water, permitting their use in high-throughput screens based on 96 well plates. Although zebrafish genomic sequencing is still not complete, this small fish has already attracted large interest because it represents an inexpensive in vitro vertebrate model. The tractability of zebrafish in small molecule screens, due to its size, transparency and permeability, has already demonstrated its potential in chemical genetics screens. In the case of one screen, small molecules were identified that could www.drugdiscoverytoday.com
57
Drug Discovery Today: Technologies | Target identification
specifically interfere with the central nervous system, cardiovascular system and ear development [11]. In another example, a screen using the zebrafish mutant gridlock, having a hypomorphic mutation in the gene hairy-related with C-terminal YRPW, hey-2, was used to identify small molecules that could suppress the gridlock phenotype. Remarkably, the gridlock defect is reminiscent of human congenital aorta deformities. This phenotype-driven, whole organism screen identified small chemical compounds that were able to rescue the lack of blood circulation to the tail of the embryo. The possibility to model genetic diseases in zebrafish, combined with the chemical suppressor screen, should allow the identification of novel chemical entities that could be relevant for the treatment of human genetic diseases [12].
The mouse as a model organism Although more costly and genetically complex than lower organisms, the predictive potential of the domestic mouse has made it a sine qua non in modern drug discovery. The close homology between the human and murine genome has meant that disruptions in certain pathways in the mouse result in phenotypes that closely correspond to human diseases having similar genetic perturbations. The development of molecular techniques has provided the opportunity to uncover the genetic basis of diseases observed in mice, driving a ‘phenotype-to-genotype’ discovery strategy. In an example of this approach, two mutant strains of mice were identified by virtue of a markedly obese phenotype. It was later revealed through positional cloning that one strain contained a mutation in a gene coding for a novel factor now known as leptin and the other had a mutation in the gene for the leptin receptor [13]. This discovery has led to a new understanding of the biochemical pathways in obesity and to the identification of new potential targets for therapeutic intervention. As new molecular genetic techniques have developed, it is now possible to insert new genes into the murine genome to study their function. Hyper-expressing, hyperactive and even human versions of genes have been integrated into the genome of these transgenic mice in what has become a paradigm shift toward a ‘genotype-to-phenotype’ strategy. The discovery that homologous recombination of an endogenous gene with an inactive version in murine embryonic stem cells can lead to the selective loss of gene function has provided a powerful new tool for drug discovery. The creation of ‘knockout’ mice from embryonic stem cells has become a standard method for discovering the function of genes. Moreover, marker genes might be inserted into the target gene that not only disrupt normal gene function but also allow analysis of tissue expression patterns. In another modification of the technique, ‘knockin’ mice can be generated in which the endogenous gene is replaced by a different version, such as an allele associated with human disease. As the technologies for 58
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Vol. 1, No. 1 2004
generating knockout mice have advanced, permitting the high-throughput generation of gene-targeted mice, a goal has emerged from both the academic and industrial sectors to create a knockout mouse for every gene. This would provide a unique opportunity to discover the function of all genes and identify new drug targets. As genes for certain potentially druggable target proteins might be necessary for embryonic viability, alternative approaches to the classical knockout strategies need to be employed to investigate gene function in the adult. Conditional knockout mice have been generated by flanking the target gene with locus of crossover sites, loxP sites, which are recognized and excised, together with the intervening DNA sequences, by the cyclization recombinase enzyme, Cre [14]. Finally, the recent discovery that RNA interference (RNAi) strategies can also silence gene expression in mammalian cells, has proven invaluable in understanding the function of genes. The most significant hurdle in utilizing this technology in drug discovery models has been the efficient delivery of interfering RNAs in vivo. Recently, it has been demonstrated that lentiviral vector-based delivery of short hairpin RNAs (shRNA) into embryonic cells can result in transgenic mice that are deficient in gene function [15]. With these new tools available, it is now possible to identify the role of specific genes in disease and gauge the impact of targeting these genes in the treatment of these diseases
Conclusions Because many human disease-associated genes are conserved through evolution, simple model organisms are probable to play a key role in drug discovery and development in the postgenomic era. To effectively use model organisms to discover drug mechanisms of action or to identify novel targets, the physiology and specific molecular genetic technologies available for each system must be considered. For example, numerous molecular genetic techniques are available for using yeast as a screening tool, allowing the design of sensitive screens using engineered transcription factors or reconstituted biochemical pathways. The ease of use of yeast for screening is underscored by the success and widespread use of two hybrid screening technology. Several mammalian signaling pathways have been reconstituted in this naı¨ve cellular milieu, and chemicals or genes directly affecting a specific pathway can be effectively screened. In general, yeast should be considered a good organism with which to screen direct biochemical interactions without the ‘‘noise’’ of a more complex cellular systems. In turn, the simplicity of the system is also one of its limitations, and the single cell lifestyle of yeast precludes the analysis of more complex processes typical of higher organisms. The use of Drosophila and C. elegans to drive drug discovery has just begun, but based on their past contribution to biology, the future of this model in pharmaceutical research
Vol. 1, No. 1 2004
is bright. Both models have established genetic and genomic techniques that are supported by well-defined biology. For both systems, the complete genomic sequence is available, and both models are amenable to large-scale screening. The ease of scoring phenotype of large numbers of genetic and chemically-induced mutants permits the screening of virtually the complete genome and the identification of all possible components in a given biochemical pathway. The use of these organisms should, in the near future, allow the identification of novel targets relevant for human diseases. The zebrafish is the most novel of the organisms that has been developed for studying complex processes such as organogenesis and embryonic development. Because of its novelty, it lacks the vast array of molecular genetic tools available for other systems. In fact, its genome sequence is still being identified and gene-targeted knockout strategies have not yet been developed. Although the zebrafish has been refractory to gene knock-down using RNAi, alternative methods using antisense derivatives have been successful. In addition, a variety of technologies, such chemical mutagenesis and transgenesis have been successfully developed and widely used to analyze gene function in this organism. Although limited by throughput and cost, the mouse is currently the most widely used organism in drug discovery and development. The complexities that lead to these limitations are the very same ones that make the mouse an ideal model in other respects. Concepts of toxicity and therapeutic window resulting from the non-selective nature of many drugs can best be appreciated in such complex systems. Likewise, the common pathological basis of disease in mice and humans has permitted the assessment of drug activity in rodent models that predict efficacy in the clinic. Given the breadth of the drug discovery effort, each of these model organisms will probably play an important role
Drug Discovery Today: Technologies | Target identification
in the identification of today’s targets and tomorrow’s drugs. As drug targets continue to slowly give up their secrets, the availability of diverse model systems will continue to serve this effort well.
References 1 Lander, E.S. et al. (2001) Initial sequencing and analysis of the human genome. Nature 409, 860–921 2 Ray, B.L. and Sturgill, T. (1987) Rapid stimulation by insulin of a serine/ threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubuleassociated protein 2 in vitro. Proc. Natl. Acad. Sci. USA 84, 1502–1506 3 Teague, A.M. et al. (1986) Nucleotide sequence of the yeast regulatory gene STE7 predicts a protein homologous to protein kinases. Proc. Natl. Acad. Sci. USA 83, 7371–7375 4 Delaney, A.M. et al. (2002) Identification of a novel mitogen-activated protein kinase kinase activation domain recognized by the inhibitor PD 184352. Mol. Cell. Biol. 22, 7593–7602 5 Sebolt-Leopold, J.S. et al. (1999) Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat. Med. 5, 810–816 6 Fire, A. et al. (1998) Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature 391, 806–811 7 Choy, R.K. and Thomas, J.H. (1999) Fluoxetine-resistant mutants in C. elegans define a novel family of transmembrane proteins. Mol. Cell. 4, 143–152 8 Clemens, J.C. et al. (2000) Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97, 6499–6503 9 Kiger, A.A. et al. (2003) A functional genomic analysis of cell morphology using RNA interference. J. Biol. 2, 27 10 Aparicio, S. et al. (2002) Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297, 1301–1310 11 Peterson, R.T. et al. (2000) Small molecule developmental screens reveal the logic and timing of vertebrate development. Proc. Natl. Acad. Sci. USA 97, 12965–12969 12 Peterson, R.T. et al. (2004) Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation. Nat. Biotechnol. 22, 595–599 13 Zhang, Y. et al. (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425–432 14 Kuhn, R. et al. (1995) Inducible gene targeting in mice. Science 269, 1427–1429 15 Rubinson, D.A. et al. (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406
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59
Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Target identification
Model organisms and target discovery Marco Muda*, Sean McKenna Serono Reproductive Biology Institute Inc., One Technology Place, Rockland MA 02370, USA
The wealth of information harvested from full genomic sequencing projects has not generated a parallel increase in the number of novel targets for therapeutic intervention. Several pharmaceutical companies have realized that novel drug targets can be identified and validated using simple model organisms. After decades of service in basic research laboratories, yeasts, worms, flies, fishes, and mice are now the cornerstones of modern drug discovery programs.
Introduction To answer fundamental questions about complex biological processes, biologists have tried to identify and develop simple and tractable model systems. Breakthrough discoveries made by studying prokaryotes paved the way to the studies of more complex organisms like yeasts, nematodes, flies, fishes, mice, and humans. Somewhat paradoxically, little use has been made of these same organisms in the development of drugs. With new light shed from full genome sequencing projects, the use of model organism in drug discovery is getting a new boost. Indeed, one of the main outcomes of the genomic sequencing projects is the recognition that many human genes, including those associated with diseases, are conserved in evolution from yeast to man. More specifically, full genomic sequence comparisons have in fact revealed that 46% of Saccharomyces cerevisiae, 43% of Caenorhabditis elegans, 61% of Drosophila melanogaster, 80% of zebrafish, and up to 97% mouse genes are similar to human genes [1]. This should not be astonishing because biological research had already demonstrated that many of the mechanisms developed by prokaryotic and eukaryotic cells to use energy, regulate gene *Corresponding author. (M. Muda) [email protected] 1740-6749/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2004.08.001
Section Editors: Wolfgang Fischer, Rob Hooft, Michael Walker For ethical and practical reasons, we use model organisms in the search for drugs that are, in the end, to be used in humans. Which model organism to choose is a trade-off between similarity to ourselves (chimps, other primates), versus ease of use and manipulation (Escherichia coli). Between these two extreme examples are choices that prioritize being a simple, fast-breeding eukaryote (yeast), insect (body plan/flies), vertebrate development (nematode or zebrafish) or small mammal (mouse). It is important to realize that the basis for these choices evolves with our understanding of biology and disease; for instance, bacteria were studied in the 1960s and 1970s to understand replication, transcription, and translation, but the trend has since moved towards more complex models. This review presents an overview of the major model organisms that are in vogue today, with an emphasis on how they are used to discover conserved signaling mechanisms.
expression and respond to environmental challenges utilize similar basic biochemical processes. Nature has invented a limited number of biochemical mechanisms regulating cellular processes and these same mechanisms have been used, reused, and modified during evolution. The concept of biochemical conservation is not novel and, in fact, the French Nobel laureate Jacques Monod remarked in 1965, ‘‘What is true for E. coli is true for an elephant.’’ This notion has permeated and was the foundation of molecular biology research that began in the first half of the previous century. The use of simple model organisms to dissect complex biological processes has permitted biology to advance at an impressive pace, and the knowledge generated by integrating genetic and biochemical studies has allowed scientists to begin to understand the molecular basis of complex diseases such as cancer and diabetes. Several pharmaceutical companies have started developing research www.drugdiscoverytoday.com
55
Drug Discovery Today: Technologies | Target identification
Vol. 1, No. 1 2004
Table 1. Model organism in drug discovery comparison Common name (scientific name)
Baker’s yeast (S. cerevisiae)
Round worm (C. elegans)
Fruit fly (D. melanogaster)
Zebrafish (Danio Rerio)
Mouse (Mus musculus)
Genome size (genes number)
6000
20,000
14,000
In progress
35,000
Whole genome sequence
Yes
Yes
Yes
In progress
Yes
Generation time
2h
3 days
10 days
3 months
3 months
HTS whole animal assay 96 well format
Yes
Yes
Yes, larva stage
Yes, larva stage
No
Gene silencing
Homologus recombination Transposon
RNAi
Homologus recombination Transposon RNAi
Morpholino
Homologus recombination
Homologous recombination = reaction between two DNA molecules used to modify or disrupt the sequence of a gene at its specific location in the genome. Transposon = mobile genetic element used to disrupt or alter gene expressions.
programs that use simple organisms to identify and validate drug targets. There are five very popular model organisms that can be used to discover novel drug targets, and each has a distinct collection of molecular tools (see Table 1 below). The complete genomic sequence is known for most of them, and using specific molecular genetic methods a genomic wide screen can be designed to identify novel genes relevant for human diseases. Because of their small size, short generation time, high fecundity and cost effective maintenance, certain model organisms can also be used for high-throughput screens (HTS) of small molecules. In fact, in many cases, standard microtiter plates (96 wells) can be used for whole animal assays to assess toxicity, identify novel therapeutic compounds, and unravel drug mechanisms of action. This review will focus on the use of model organisms, giving some examples demonstrating the use of model systems for the dissection of biological processes relevant for human diseases. Furthermore, when possible, it will illustrate some recent reports revealing how model organisms can be used to advance drug discovery.
Baker’s yeast as a model organism S. cerevisiae (Baker’s yeast) has been extensively used to study the molecular basis of cell division and gene regulation in eukaryotic cells. S. cerevisiae has been instrumental in the development of technologies that allow the identification and dissection of protein–protein interactions using technologies such as two-hybrid screens. Moreover, the study of yeast biology has also been instrumental in understanding signaling pathways such as G-protein coupled receptors and protein kinases, both of which are important targets in drug development. One kinase-based signaling cascade that in recent years has benefited from basic discoveries in yeast is the mitogenactivated protein kinase extracellular or receptor-stimulated 56
www.drugdiscoverytoday.com
kinase, MAPK/ERK. The first member of this family was isolated from insulin-treated adypocytes [2]. MAPKs are activated by MAPK kinases (MAPKK) which themselves are dependent upon a further upstream kinase, MAPKKK. The first kinase responsible for MAPK activation was earlier identified as a mutant in the mating response of S. cerevisiae STE7 [3]. The MAPK cassette, composed of three kinases sequentially activated, is now recognized as a universal signaling module vital for cellular adaptation, proliferation and differentiation. Elements of the MAPK pathways are now recognized targets for drug development for treating cancer, inflammation and immune diseases. Not only has S. cerevisiae been important for the early fundamental discoveries in the MAPK cascade; recently, scientists at Pfizer have used S. cerevisiae to clarify the mechanism of action of PD 184353, a small-molecule inhibitor of MAPK/ERK kinase, MEK, the human MAPKK [4]. MEK inhibitors are promising novel compounds that are in development for the treatment of several types of tumors and have a demonstrated ability to block growth of colon cancer in mice [5]. To identify the domain recognized by the inhibitor PD 184353, Delaney et al. reconstituted the human MAPK signaling cascade in yeast, conferring the ability to grow in the absence of one amino acid. A library of mutated MEK was generated and screened for mutations that conferred resistance to the inhibitor. Several mutants were identified and this information, together with structural modeling, identified a novel hydrophobic pocket that is likely to be involved in the binding of PD 184353.
A worm as a model organism In the early 1960s, after breakthrough discoveries in molecular biology had solved fundamental questions in biology, Sydney Brenner proposed to use the nematode C. elegans to begin attacking the problems of cellular development. C. elegans was chosen because the small, transparent nematode
Vol. 1, No. 1 2004
was amenable to microscopic visualization to follow the development and fate of all the 1090 cells that are generated during early development. During C. elegans development, 109 cells are lost by programmed cell death or apoptosis. This characteristic made C. elegans a useful genetic model for the identification of the genes involved in the regulation of programmed cell death. In cells destined to die, the level of the egg-laying abnormal-1 protein, EGL-1, is increased. EGL-1 interacts with a protein complex composed of cell death abnormal-9, CED-9 (similar to human B-cell lymphoma-2, BCL-2) and CED-4, releasing CED-4 (similar to human apoptotic protease activating factor-1, Apaf-1) that in turn activates CED-3 (similar to human Caspases). As in humans, C. elegans programmed cell death is a major feature of the normal development of the nervous system. Importantly, uncontrolled cell death is thought to be the cause of several neurological disorders such as stroke and neurodegenerative diseases. Underscoring the importance of groundbreaking work in C. elegans on the molecular basis of development and apoptosis, Sydney Brenner, Robert H. Horvitz, and John E. Sulston were awarded the Nobel Prize in Physiology or Medicine in 2002. In addition to its fundamental contribution illuminating the process of programmed cell death, C. elegans has been instrumental in the discovery of double-stranded RNA interference (RNAi), which has now become a widely used method in gene functional studies from fly to human. Discovery of this phenomenon came from antisense RNA experiments, where it was observed that both sense and antisense RNAs were effective at producing pheno-copies of genetic loss-offunction mutations. It was later determined that combination of sense and antisense strands, producing dsRNA, were necessary for potent and specific knock-down of protein expression [6]. C. elegans has just started to be explored for drug development, but it has already shown potential in the identification of novel drug mechanisms of action. A genetic screen based on C. elegans has demonstrated that Prozac might have additional in vivo effects distinct from the well-accepted role in blocking serotonin re-uptake. It remains to be seen whether other anti-depressants that block serotonin reuptake have similar unexpected activities that might have implications in the clinical side-effect profiles of this class of drug [7].
The fruit fly as a model organism Due to ease of culturing and its high fecundity, D. melanogaster has been the workhorse of geneticists since T.H. Morgan’s seminal work at the beginning of 1900. Many of the foundations of modern biology have been inspired by the discoveries made with this small insect. Indeed, in 1995 Christiane Nusslein-Volhard and Eric Wiechaus were awarded the Nobel Prize for their groundbreaking research
Drug Discovery Today: Technologies | Target identification
aimed at the identification of the genes involved in embryonic development. In recent years, several original discoveries made during studies in Drosophila have shed light on complex human diseases such as cancer and diabetes. In fact, study of the oncogene ras (identified from rats with sarcoma) in this model has identified novel genes that modulate the same signaling pathway in man. Mutant forms of ras were among the first oncogenes identified; three distinct ras genes named H-ras, K-ras and N-ras are encoded in the human genome. Ras proteins belong to a large family of small GTP-binding proteins that regulate diverse cellular functions. Normal Ras proteins can bind either GDP or GTP, and mutations of Ras that lock the protein conformation in the GTP-bound state are frequently found in human cancer. Ras has been shown to associate and activate Ras activated factor, Raf, a MAPKKK upstream of MEK (see above), and constitutive activation of this pathway is sufficient to drive cell transformation and tumor formation. Importantly, the guanine nucleotide exchange factor responsible for the exchange of GDP to GTP on Ras, named Son-of-Sevenless (Sos), was identified in a Drosophila genetic screen aimed at identifying genes involved in the differentiation of photoreceptor cells. As in mammalian systems, Sos acts downstream of receptor tyrosine kinases, whereas sevenless is the gene encoding the receptor essential for photoreceptor differentiation. Drosophila has the additional advantage that cell lines have been established that can be used to perform biochemical experiments that complement genetic studies. Remarkably, RNAi has been extensively implemented using Drosophila cell lines and this powerful yet simple technology has been used to dissect signaling pathways and to identify novel genes regulating cytoskeleton dynamics [8,9].
A fish as a model organism Because of its relatively small and compact genome size, Fugu rubripes, commonly known as puffer fish, made an attractive model for comparative genomic studies [10]. However, because of its relative large size and slow reproductive rate, Fugu has not yet been widely used as a tool in drug discovery. By contrast, Danio rerio (zebrafish) has many features that render it an attractive alternative to other model organisms, including high fecundity, extra uterine development and transparency of the embryo allowing direct observation of internal organs during early stages of development. Zebrafish larvae can live in less than 100 ml of water, permitting their use in high-throughput screens based on 96 well plates. Although zebrafish genomic sequencing is still not complete, this small fish has already attracted large interest because it represents an inexpensive in vitro vertebrate model. The tractability of zebrafish in small molecule screens, due to its size, transparency and permeability, has already demonstrated its potential in chemical genetics screens. In the case of one screen, small molecules were identified that could www.drugdiscoverytoday.com
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specifically interfere with the central nervous system, cardiovascular system and ear development [11]. In another example, a screen using the zebrafish mutant gridlock, having a hypomorphic mutation in the gene hairy-related with C-terminal YRPW, hey-2, was used to identify small molecules that could suppress the gridlock phenotype. Remarkably, the gridlock defect is reminiscent of human congenital aorta deformities. This phenotype-driven, whole organism screen identified small chemical compounds that were able to rescue the lack of blood circulation to the tail of the embryo. The possibility to model genetic diseases in zebrafish, combined with the chemical suppressor screen, should allow the identification of novel chemical entities that could be relevant for the treatment of human genetic diseases [12].
The mouse as a model organism Although more costly and genetically complex than lower organisms, the predictive potential of the domestic mouse has made it a sine qua non in modern drug discovery. The close homology between the human and murine genome has meant that disruptions in certain pathways in the mouse result in phenotypes that closely correspond to human diseases having similar genetic perturbations. The development of molecular techniques has provided the opportunity to uncover the genetic basis of diseases observed in mice, driving a ‘phenotype-to-genotype’ discovery strategy. In an example of this approach, two mutant strains of mice were identified by virtue of a markedly obese phenotype. It was later revealed through positional cloning that one strain contained a mutation in a gene coding for a novel factor now known as leptin and the other had a mutation in the gene for the leptin receptor [13]. This discovery has led to a new understanding of the biochemical pathways in obesity and to the identification of new potential targets for therapeutic intervention. As new molecular genetic techniques have developed, it is now possible to insert new genes into the murine genome to study their function. Hyper-expressing, hyperactive and even human versions of genes have been integrated into the genome of these transgenic mice in what has become a paradigm shift toward a ‘genotype-to-phenotype’ strategy. The discovery that homologous recombination of an endogenous gene with an inactive version in murine embryonic stem cells can lead to the selective loss of gene function has provided a powerful new tool for drug discovery. The creation of ‘knockout’ mice from embryonic stem cells has become a standard method for discovering the function of genes. Moreover, marker genes might be inserted into the target gene that not only disrupt normal gene function but also allow analysis of tissue expression patterns. In another modification of the technique, ‘knockin’ mice can be generated in which the endogenous gene is replaced by a different version, such as an allele associated with human disease. As the technologies for 58
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generating knockout mice have advanced, permitting the high-throughput generation of gene-targeted mice, a goal has emerged from both the academic and industrial sectors to create a knockout mouse for every gene. This would provide a unique opportunity to discover the function of all genes and identify new drug targets. As genes for certain potentially druggable target proteins might be necessary for embryonic viability, alternative approaches to the classical knockout strategies need to be employed to investigate gene function in the adult. Conditional knockout mice have been generated by flanking the target gene with locus of crossover sites, loxP sites, which are recognized and excised, together with the intervening DNA sequences, by the cyclization recombinase enzyme, Cre [14]. Finally, the recent discovery that RNA interference (RNAi) strategies can also silence gene expression in mammalian cells, has proven invaluable in understanding the function of genes. The most significant hurdle in utilizing this technology in drug discovery models has been the efficient delivery of interfering RNAs in vivo. Recently, it has been demonstrated that lentiviral vector-based delivery of short hairpin RNAs (shRNA) into embryonic cells can result in transgenic mice that are deficient in gene function [15]. With these new tools available, it is now possible to identify the role of specific genes in disease and gauge the impact of targeting these genes in the treatment of these diseases
Conclusions Because many human disease-associated genes are conserved through evolution, simple model organisms are probable to play a key role in drug discovery and development in the postgenomic era. To effectively use model organisms to discover drug mechanisms of action or to identify novel targets, the physiology and specific molecular genetic technologies available for each system must be considered. For example, numerous molecular genetic techniques are available for using yeast as a screening tool, allowing the design of sensitive screens using engineered transcription factors or reconstituted biochemical pathways. The ease of use of yeast for screening is underscored by the success and widespread use of two hybrid screening technology. Several mammalian signaling pathways have been reconstituted in this naı¨ve cellular milieu, and chemicals or genes directly affecting a specific pathway can be effectively screened. In general, yeast should be considered a good organism with which to screen direct biochemical interactions without the ‘‘noise’’ of a more complex cellular systems. In turn, the simplicity of the system is also one of its limitations, and the single cell lifestyle of yeast precludes the analysis of more complex processes typical of higher organisms. The use of Drosophila and C. elegans to drive drug discovery has just begun, but based on their past contribution to biology, the future of this model in pharmaceutical research
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is bright. Both models have established genetic and genomic techniques that are supported by well-defined biology. For both systems, the complete genomic sequence is available, and both models are amenable to large-scale screening. The ease of scoring phenotype of large numbers of genetic and chemically-induced mutants permits the screening of virtually the complete genome and the identification of all possible components in a given biochemical pathway. The use of these organisms should, in the near future, allow the identification of novel targets relevant for human diseases. The zebrafish is the most novel of the organisms that has been developed for studying complex processes such as organogenesis and embryonic development. Because of its novelty, it lacks the vast array of molecular genetic tools available for other systems. In fact, its genome sequence is still being identified and gene-targeted knockout strategies have not yet been developed. Although the zebrafish has been refractory to gene knock-down using RNAi, alternative methods using antisense derivatives have been successful. In addition, a variety of technologies, such chemical mutagenesis and transgenesis have been successfully developed and widely used to analyze gene function in this organism. Although limited by throughput and cost, the mouse is currently the most widely used organism in drug discovery and development. The complexities that lead to these limitations are the very same ones that make the mouse an ideal model in other respects. Concepts of toxicity and therapeutic window resulting from the non-selective nature of many drugs can best be appreciated in such complex systems. Likewise, the common pathological basis of disease in mice and humans has permitted the assessment of drug activity in rodent models that predict efficacy in the clinic. Given the breadth of the drug discovery effort, each of these model organisms will probably play an important role
Drug Discovery Today: Technologies | Target identification
in the identification of today’s targets and tomorrow’s drugs. As drug targets continue to slowly give up their secrets, the availability of diverse model systems will continue to serve this effort well.
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