Drug Discovery Today: Technologies
Vol. 1, No. 2 2004
Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Target validation
Developments in modulating protein function for effective target validation Leodevico L. Ilag1 Xerion Pharmaceuticals AG, Sauerbruchstrasse 50, 81377 Munich, Germany
The targets of more than 95% of clinically approved drugs are proteins. Thus, the plethora of targets derived from genomics and proteomics efforts must be validated at the protein level. However, most of the preferred target validation technologies are gene- or transcript-based. Protein-based or proteinetic approaches, which are more relevant to determine target druggability, are now emerging. Introduction Genomics has thus, so far, failed to deliver on its promise to increase drug discovery efficiency because of its complacency on target druggability (see Glossary). As the bottleneck has shifted to target validation (see Glossary), some parallel mistakes from genomics are in the process of being repeated. Since proteins represent more than 95% of druggable targets, it is imperative that validation is performed at the protein level. Yet, most of target validation is still done at the nucleotide level, as evidenced by the popularity of RNAi (see Glossary). This does not imply that nucleotide-based technologies are irrelevant, but they are complementary towards the eventual validation by direct modulation of protein function. This mini-review discusses new developments on methods that directly modulate protein function. One unifying characteristic of these proteinetic (see Glossary) methods is the increase in efficiency and the ability to conditionally inhibit protein function permitting precise modulation of protein function. E-mail address: [email protected]. URL: http://www.xerion-pharma.com/. 1
Present address: Cryptome Pharmaceuticals Ltd, Melbourne, Australia.
1740-6749/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2004.08.006
Section Editors: Luis Menandez-Arias, Pierre Chatelain, Bernard Masereel Technologies focusing on the direct modulation of protein activity, such as chromophore-assisted laser inactivation, provide a complementary approach to gene knockout and other validation techniques targeting gene expression. Protein inactivation techniques show high spatial and temporal resolution and can be used to validate novel targets (i.e. a single domain of a protein), missed by other inactivation strategies. This review was selected because protein inactivation technologies are expected to play a key role in the development of functional proteomics, through the identification of multiple interactions between gene products, and addressing the role of proteins in complex cellular pathways.
Key technologies CALI and FALI Antibodies have been often used for protein inactivation studies but the efficiency of generating a neutralizing antibody against a particular target is low. Thus, chromophoreassisted laser inactivation (CALI) was developed as a tool to increase the efficiency of antibodies and other non-neutralizing binding molecules in exploring protein function in cell and animal systems that were not amenable to genetic manipulation [1]. Briefly, a chromophore with special photochemical properties is linked to a ligand such as an antibody, peptide or small molecule. Laser or incoherent light induces a photochemical reaction that generates reactive oxygen species (hydroxyl radicals or singlet oxygen) that diffuses within a limited range leading to modification of amino acids of the target protein. If these modified amino acids play a role in the function of the protein, the particular protein domain is inactivated. Thus, it efficiently converts a non-functional ligand into conditional functional inhibitors. CALI has been www.drugdiscoverytoday.com
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Vol. 1, No. 2 2004
Glossary Druggability: refers to a targets property of being amenable to the design or isolation of a pharmaceutical modulator (agonist or antagonist) against it. ‘‘Forward’’ and ‘‘reverse’’: in both genetics and proteinetics, ‘‘forward’’ approach refers to the use of a selection or screen to identify the variants of interest from a diverse population followed by the determination of the responsible genotype. In the ‘‘reverse’’ approach, the genotype is predetermined or modified and the resulting or appropriate phenotype is directly evaluated. Proteinetics: is a newly coined word to differentiate the direct modulation of function at the protein level compared to genetics that has been associated to the manipulation of the genetic material, mainly nucleic acids. Genetics with associated techniques and technologies has significantly contributed to the understanding of protein function. However, a differentiation is necessary as proteins have multiple functions and properties and that modulation at the genetic level does not often directly translate to modulation at the protein level, which is important in how a pharmacological agent works RNAi or RNA interference: is a process involving the specific degradation of transcripts of particular genes through the use of short double-stranded RNA that represent a segment of the target transcript.
successfully applied in more than 50 proteins (both intracellular and extracellular) in several in vitro cellular and animal model systems to elucidate the functional role of proteins (reviewed in [2]) (Fig. 1). Malachite green was the original chromophore used in ˚ from CALI and has an estimated modification radius of 15 A the chromophore and requires a pulsed laser. However, the poor solubility, cross-linking properties of malachite green to ligands, and requirement of an expensive pulsed laser system have been cumbersome. Thus, it was a boon when fluorescein, a fluorophore, was discovered to provide the same effects as CALI using a continuous wave laser [3]. The subsequent discovery that incoherent light can produce similar effects further opened the possibility for high-throughput multiplex applications. These developments led to fluorophore-assisted laser inactivation (FALI), which is now the preferred choice. With fluorescein, the effective radius of ˚ , making it still within the range protein modification is 40 A of inhibiting specific protein domains [4,5]. Most applications of CALI/FALI have been based on a hypothesis-driven approach involving inactivating a known protein and trying to validate its role in a particular biological process or pathway. The application of FALI in a forward genetic-like screen was recently demonstrated [6]. In a functional proteomic screen of the extracellular proteome of a tumor cell for proteins involved in tumor cell invasion, FALI was used in combination with mass spectrometry and antibody technologies to reveal a novel essential role for extracellular hsp90a, a target for cancer therapy. The findings suggest new therapeutic approaches of inhibiting progression of cancer that may increase the efficacy and safety of the current generation of hsp90 inhibitors. Such discovery of 114
www.drugdiscoverytoday.com
Figure 1. Principle of chromophore-assisted laser inactivation (CALI). (a) A target protein is (b) complexed with a ligand (yellow; e.g. antibody, antibody fragments such as scFv and Fab, peptide, nucleic acid aptamers, small molecules) labeled with dye molecules (green; e.g. malachite green, fluorescein). The complex is irradiated with laser or incoherent light (red lightning) leading to (c) the generation of reactive species (concentric circles) that travel a short distance resulting in (d) modifications (black) of nearby amino acids. If the modified amino acids are responsible for a function, that particular functional protein domain will be inactivated, leaving the rest of the other functional domains intact and remain functional. In genetically targeted CALI or FALI, the chromophore is directly attached to the target protein. This can be accomplished using GFP genetically fused to the target protein, whereby GFP serves as the chromophore at the same time. Alternatively, a tetracysteine tag genetically fused to the target protein will specifically capture the chromophore (ReASH or FlASH) to facilitate CALI.
hsp90a’s extracellular location including its functional interaction with matrix metalloprotease 2 would have not been possible with genetic or RNAi approaches. This further highlights the importance of validating target function at the protein level (see Outstanding issues). Both CALI and FALI provide high spatial resolution inactivation of protein domains mimicking how a pharmacological agent will affect a target protein. However, one disadvantage with these methods is when the target protein turnover is high because of continuous synthesis of new proteins by the cellular machinery leading to a compensatory effect or negative CALI/FALI effect. For intracellular targets, the ligand should be delivered in sufficient quantities into the cell. Establishing controls to demonstrate that the ligand delivered into the cell is binding to the target protein intracellularly remains a challenge. Thus, negative results from CALI and FALI are not easily subject to interpretation.
Vol. 1, No. 2 2004
Genetically-targeted CALI Two recent approaches have addressed the possibility of efficiently targeting targets with CALI or FALI, circumventing the use of ligands specific to the target protein. At the same time, it addresses the limitations of CALI/FALI against intracellular targets. Instead of using a ligand that can be linked to a chromophore or fluorophore, a tag with specificity to chromophores or a fluorescent protein is directly fused to the target protein as a genetic fusion. Thus, the translated protein would have a high probability of being affected by CALI or FALI. In the first setting, a fluorescent protein (e.g. green fluorescent protein (GFP)) is genetically fused to a target protein. Rajfur et al. [7] used enhanced green fluorescent proteins (EGFP) for CALI towards understanding cytoskeletal structure by modulating the function of a-actinin through laser irradiation of EGFP-a-actinin expressed in Swiss 3T3 fibroblasts, leading to the detachment of stress fibers from focal adhesions (FAs). The CALI of EGFP–FAK (focal adhesion kinase) was spatially resolved in that only the kinase activity of FAK was inactivated, which is consistent with other experimental results. Although these studies have provided significant results, the efficiency of inactivation is not satisfactory compared to the use of organic dyes and may be limited by the fusion of a significantly large protein domain into the target protein that can severely affect protein function. In a second setting, to take advantage of the higher efficiency of organic dye molecules for generating reactive species instead of GFP for in vivo applications, a transgenically encoded tag was used that comprised a tetracysteine motif that specifically binds a modified form of fluorescein (FlAsH) and a membrane permeant, red biarsenical dye (ReAsH), calling the techniques FlAsH-FALI [8] and ReAsH-mediated CALI [9], respectively. FlAsH and ReAsH are only fluorescent upon binding to the tetracysteine motif. FlAsH-FALI and ReAsH-mediated CALI is specific and proportional to the duration of illumination and followed first-order kinetics. In an application of FlAsH-FALI, the authors engineered into synaptotagmin I (Syt I), a tetracysteine motif that binds the membrane-permeable, non-fluorescent fluorescein derivative 40 50 -bis (1,3,2-dithioarsolan-2yl) fluorescein (FlAsH) [8]. The engineered synaptotagmin I was introduced into Drosophila and was functionally expressed followed by the introduction of FlAsH to inactivate it. ReAsH-mediated CALI was successfully applied to inactivate connexin43 and a1C L-type calcium channels [9]. The authors further showed that ReAsH was much more efficient than FlAsH or GFP in generating reactive oxygen species leading to better inactivation. In addition to being more efficient than EGFP, the tetracysteine motif is only a few amino acids compared to GFP, making it more tolerable for protein fusions. However, the use of the teteracysteine motif might not be applicable
Drug Discovery Today: Technologies | Target validation
to proteins that are transported into the endoplasmic reticulum where the oxidizing environment might disrupt the motif. FlAsH-FALI and ReAsH-mediated CALI are analogous to the analog sensitive enzyme alleles (ASEA) methods described below but may be more versatile since it does not require prior knowledge or bias towards a functional site of the target molecule and the ligand does not have to be modified. Conceptually this elegant approach can be applied in a forward (see Glossary) genetic screen for tagging different genomics targets with the tetracysteine motif and randomly performing FlAsH-FALI and ReASH mediated CALI with the appropriate phenotypic screens.
Analog sensitive enzyme alleles The fundamental concept of ASEA is based on the modification of a ligand (e.g. small molecule) followed by the complementary modification of the receptor (e.g. target protein) facilitating specific interaction between the ligand and receptor [10]. The modified ligand is referred to as orthogonal in normal cells because it cannot interact with its receptor and preferably any other target protein in the cell. The new target receptor protein is engineered to accept the newly modified ligand. The newly engineered protein retains its normal physiological functions except for its new ability to interact with the modified ligand. More recent ASEA approaches have been applied to kinases, which regulate cellular processes and represent about 2% of all human genes. A set of analogsensitive kinase alleles (ASKA) and small molecule scaffolds were developed leading to an approach that can systematically probe the physiological roles of the different kinase families without the need for specific engineering of each small molecule and protein [11]. The key to ASKA is the ability to create a subtle yet distinct discriminant in one kinase, distinguishing it from the rest of the other kinases by modifying the ATP binding site. This mutation leads to ‘‘normal’’ functioning kinase with preference for a modified ATP analog. The amino acid residue targeted for mutation lies in the ATP binding site and is conserved among all kinases, making the modification of target kinases possible without knowledge of their three-dimensional structures. This approach has been successfully applied to a variety of kinases (reviewed in [12]). A most recent application that highlights the technology was demonstrated to a complex immunological system whereby novel p56 (LcK) ASKAs were used to show dose-dependent correlation of thymocyte development with varying Lck activity [13]. The ASEA principle has also been extended beyond kinases. A recent class of analogspecific enzyme alleles has been directed against protein methyltransferases [14]. The application of ASEA in vivo is a powerful demonstration of the technology’s potential. This was demonstrated in defining the role of myosin-1c (also known as myosin-1b) www.drugdiscoverytoday.com
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Vol. 1, No. 2 2004
in the adaptation of sensory hair cells among the 40 different myosins implicated in the process [15]. ASEA circumvents the generation of multiple lines of mice expressing different levels of transgenes. ASEA will eventually be the method of choice in validating proteins that are amenable to the principle of orthogonal chemical genetics in vivo because it directly mimics how a pharmacological agent works. However, one limitation is that only functional domains that are amenable to orthogonal chemical genetics can be manipulated and other protein domains that are not amenable to analogs will be inaccessible.
Comparison of technologies With the plethora of targets one can take a ‘‘reverse’’ or hypothesis-driven approach whereby a putative target is modulated and its association with a particular biological process or pathway is deduced from a relevant phenotypic or biochemical assay. The limitation of this approach is that one has to sort individually through the thousands of different genes or proteins against multitudes of different assays, which can be tedious. In the ‘‘forward’’ approach, once a functional hit is identified, the corresponding target responsible for the modulation of the function or phenotype is deconvoluted. The advantage of this approach is that one
can sort through the proteome rapidly for the proteins relevant to a particular disease or biological pathway. One compromise is that certain targets might be missed out by virtue of the sensitivity and efficiency of the technology to modulate the particular target. From the cellular screen it is imperative that validation continues in a relevant in vivo model (see Outstanding issues). The aforementioned technologies (for summary see Table 1) are best suited for either the ‘‘reverse’’ or ‘‘forward’’ approaches. The ASEA technology would be very valuable for analyzing different families of proteins such as kinases in vivo. One disadvantage is that modulation is limited to the catalytic sites. It is known that proteins have additional functional domains that can be as good a target site as the catalytic domains. CALI/FALI for a large number of intracellular proteins can be slow, limited by the ability to deliver significant large number of ligands into the cells. However, the use of genetically targeted CALI/FALI offers a viable alternative. For large-scale cell-based screens it is reasonable to combine these genetically targeted CALI/FALI approaches with multiplex cloning to fuse the tetracysteine tags into different sites of the targets. Alternatively, one can have a non-biased way of inserting the tetracysteine tags into the targets by coupling with the CD tagging approach [16]. Although photodynamic
Table 1. Comparison summary table Name of specific type of technology
Chromophore/ flourophore-assisted laser/light inactivation (CALI/FALI)
Genetically targeted CALI/FALI
Analog specific enzyme alleles (ASEA)
Names of specific technologies with associated companies and company websites
Xstream and XCALIbur from Xerion Pharmaceuticals (http://www.xerion-pharma.com/)
GFP-CALI (Professor Kenneth Jacobson, University of North Carolina, http://www-cellbio.med.unc.edu/ jacobson/jacobso.htm) FlAsH-FALI (Professor Graeme Davis, University of California, San Francisco, http://www.ucsf.edu/davislab/)
P-inhibitor and P-target technologies from Cellular Genomics, Inc. (http://www.cellulargenomics.com/)
CALI and FALI (Dan Jay of Tufts University) (http://www.tufts.edu/ksloan01/ DanJay.html)
ASKA (Professor Kevan Shokat, University of California, San Francisco, http://www.ucsf.edu/shokat/)
ReAsH-CALI (Professor Roger Y. Tsien, UC San Diego, http://www.tsienlab.ucsd.edu) Reagents sold by Invitrogen (http://www.invitrogen.com/) Pros
- Acute and high spatial resolution - Amenable to extracellular targets
- Acute and high spatial resolution - Amenable to intracellular targets - In vivo applications
- Specific and acute - Directly mimics a small molecule agent - In vivo applications
Cons
- Intracellular targets are challenging
- Introduction of tags might disrupt protein function and not mimic the native state
- Limited to targets with amino acids amenable to modification - In vivo applications challenging
- Limited to targets with amino acids amenable to modification - In vivo applications limited to surface area that can be exposed to light
- Limited to proteins that have catalytic sites that are amenable to the orthogonal approach - Limited to catalytic sites and not other regulatory domains
Costs
Not available
Not available
Not available
References
[1–6]
[7–9]
[10–15]
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Related articles Hardy, L.W. and Peet, N.P. (2004) The multiple orthogonal tools approach to define molecular causation in the validation of druggable targets. Drug Discov. Today 9, 117–126 Ilag, L.L. (2003) Direct methods for modulating protein function. Curr. Opin. Drug Discov. Dev. 6, 262–268 Shokat, K. and Velleca, M. (2002) Novel chemical genetic approaches to the discovery of signal transduction inhibitors. Drug Discov. Today 7, 872–879 Ilag, L.L. et al. (2002) Emerging high-throughput drug target validation technologies. Drug Discov. Today 7, S136–S142 Henning, S.W. and Beste, G. (2002) Loss-of-function strategies in drug target validation. Curr. Drug Discov. 5, 17–21
Drug Discovery Today: Technologies | Target validation
its adoption and appreciation of the need to validate targets at the protein level (see Outstanding issues).
Outstanding issues Transition from nucleotide-based validation to protein-based validation. Application of protein-based validation in more in vivo models. Use of protein-based approaches in forward proteinetic screens. Accessibility to technologies and cost.
References
therapy has been shown to work, applications of CALI or genetically targeted CALI/FALI in relevant disease animal models in vivo remains to be seen. For the forward approach, the feasibility of using FALI in a functional proteome screen has been successfully demonstrated whereby randomly selected antibodies that gave a FALI effect were used subsequently for immunoprecipitation and the cognate target proteins identified via mass spectrometry [6]. Applications to more sophisticated screens based on use of phage display for selecting on cell surface proteins and other functional assays are anticipated to follow. Conceptually, genetically targeted CALI/FALI can be used in a forward screen. One can make random insertions of the tetracysteine tag into a cell or embryos followed by addition of FlAsH or ReAsH and subsequent illumination, then read out by a particular assay. Positive hits can then be recovered by identifying which gene has been genetically linked to the tetracysteine tag.
Conclusions The convenience of RNAi technology has led to a pause in the need to validate drug targets at the protein level in cell-based screens. However, once the genomic screens with RNAi are completed, the need for validation at the protein level will once more be appreciated. The emerging protein-based approaches should take advantage of this lull period to develop into more robust and cost-effective systems. In addition, use of these proteinetic methods in a forward approach such as proteome-wide functional screens will help increase
1 Jay, D.G. (1988) Selective destruction of protein function by chromophore-assisted laser inactivation. Proc. Natl. Acad. Sci. USA 85, 5454– 5458 2 Ilag, L.L. et al. (2000) Chromophore-assisted laser inactivation (CALI) to validate drug targets and pharmacogenomic markers. Drug Dev. Res. 49, 65–73 3 Surrey, T. et al. (1998) Chromophore-assisted light inactivation and selforganization of microtubules and motors. Proc. Natl. Acad. Sci. USA 95, 4293–4298 4 Beck, S. et al. (2002) Fluorophore-assisted light inactivation (FALI): a high-throughput tool for direct validation of proteins. Proteomics 2, 247– 255 5 Rubenwolf, S. et al. (2002) Functional proteomics using chromophoreassisted laser inactivation. Proteomics 2, 241–246 6 Eustace, B.K. et al. (2004) Functional proteomic screens reveal an essential extracellular role for hsp90 in cancer cell invasiveness. Nat. Cell Biol. 6, 507–514 7 Rajfur, Z. et al. (2002) Dissecting the link between stress fibers and focal adhesions by CALI with EGFP fusion proteins. Nat. Cell Biol. 4, 286–293 8 Marek, K.W. and Davis, G.W. (2002) Transgenically encoded protein photoinactivation (FlAsH-FALI): acute inactivation of synaptotagmin I. Neuron 36, 805–813 9 Tour, O. et al. (2003) Genetically targeted chromophore-assisted light inactivation. Nat. Biotechnol. 21, 1505–1508 10 Hwang, Y.W. and Miller, D.L. (1987) A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein. J. Biol. Chem. 262, 13081–13085 11 Bishop, A.C. et al. (2000) A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401 12 Shah, K. and Shokat, K.M. (2003) A chemical genetic approach for the identification of substrates of protein kinases. Methods Mol. Biol. 233, 253–271 13 Denzel, A. et al. (2003) Cutting Edge: a chemical genetic system for the analysis of kinases regulating T cell development. J. Immunol. 171, 519– 523 14 Lin, Q. et al. (2001) Design of allele-specific protein methyltransferase inhibitors. J. Am. Chem. Soc. 123, 11608–11613 15 Holt, J.R. (2002) A chemical-genetic strategy implicates myosin-1c in adaptation by hair cells. Cell 108, 371–381 16 Jarvik, J.W. et al. (2002) In vivo functional proteomics: mammalian genome annotation using CD-tagging. Biotechniques 33, 852–854
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Vol. 1, No. 2 2004
Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Target validation
Developments in modulating protein function for effective target validation Leodevico L. Ilag1 Xerion Pharmaceuticals AG, Sauerbruchstrasse 50, 81377 Munich, Germany
The targets of more than 95% of clinically approved drugs are proteins. Thus, the plethora of targets derived from genomics and proteomics efforts must be validated at the protein level. However, most of the preferred target validation technologies are gene- or transcript-based. Protein-based or proteinetic approaches, which are more relevant to determine target druggability, are now emerging. Introduction Genomics has thus, so far, failed to deliver on its promise to increase drug discovery efficiency because of its complacency on target druggability (see Glossary). As the bottleneck has shifted to target validation (see Glossary), some parallel mistakes from genomics are in the process of being repeated. Since proteins represent more than 95% of druggable targets, it is imperative that validation is performed at the protein level. Yet, most of target validation is still done at the nucleotide level, as evidenced by the popularity of RNAi (see Glossary). This does not imply that nucleotide-based technologies are irrelevant, but they are complementary towards the eventual validation by direct modulation of protein function. This mini-review discusses new developments on methods that directly modulate protein function. One unifying characteristic of these proteinetic (see Glossary) methods is the increase in efficiency and the ability to conditionally inhibit protein function permitting precise modulation of protein function. E-mail address: [email protected]. URL: http://www.xerion-pharma.com/. 1
Present address: Cryptome Pharmaceuticals Ltd, Melbourne, Australia.
1740-6749/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2004.08.006
Section Editors: Luis Menandez-Arias, Pierre Chatelain, Bernard Masereel Technologies focusing on the direct modulation of protein activity, such as chromophore-assisted laser inactivation, provide a complementary approach to gene knockout and other validation techniques targeting gene expression. Protein inactivation techniques show high spatial and temporal resolution and can be used to validate novel targets (i.e. a single domain of a protein), missed by other inactivation strategies. This review was selected because protein inactivation technologies are expected to play a key role in the development of functional proteomics, through the identification of multiple interactions between gene products, and addressing the role of proteins in complex cellular pathways.
Key technologies CALI and FALI Antibodies have been often used for protein inactivation studies but the efficiency of generating a neutralizing antibody against a particular target is low. Thus, chromophoreassisted laser inactivation (CALI) was developed as a tool to increase the efficiency of antibodies and other non-neutralizing binding molecules in exploring protein function in cell and animal systems that were not amenable to genetic manipulation [1]. Briefly, a chromophore with special photochemical properties is linked to a ligand such as an antibody, peptide or small molecule. Laser or incoherent light induces a photochemical reaction that generates reactive oxygen species (hydroxyl radicals or singlet oxygen) that diffuses within a limited range leading to modification of amino acids of the target protein. If these modified amino acids play a role in the function of the protein, the particular protein domain is inactivated. Thus, it efficiently converts a non-functional ligand into conditional functional inhibitors. CALI has been www.drugdiscoverytoday.com
113
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Vol. 1, No. 2 2004
Glossary Druggability: refers to a targets property of being amenable to the design or isolation of a pharmaceutical modulator (agonist or antagonist) against it. ‘‘Forward’’ and ‘‘reverse’’: in both genetics and proteinetics, ‘‘forward’’ approach refers to the use of a selection or screen to identify the variants of interest from a diverse population followed by the determination of the responsible genotype. In the ‘‘reverse’’ approach, the genotype is predetermined or modified and the resulting or appropriate phenotype is directly evaluated. Proteinetics: is a newly coined word to differentiate the direct modulation of function at the protein level compared to genetics that has been associated to the manipulation of the genetic material, mainly nucleic acids. Genetics with associated techniques and technologies has significantly contributed to the understanding of protein function. However, a differentiation is necessary as proteins have multiple functions and properties and that modulation at the genetic level does not often directly translate to modulation at the protein level, which is important in how a pharmacological agent works RNAi or RNA interference: is a process involving the specific degradation of transcripts of particular genes through the use of short double-stranded RNA that represent a segment of the target transcript.
successfully applied in more than 50 proteins (both intracellular and extracellular) in several in vitro cellular and animal model systems to elucidate the functional role of proteins (reviewed in [2]) (Fig. 1). Malachite green was the original chromophore used in ˚ from CALI and has an estimated modification radius of 15 A the chromophore and requires a pulsed laser. However, the poor solubility, cross-linking properties of malachite green to ligands, and requirement of an expensive pulsed laser system have been cumbersome. Thus, it was a boon when fluorescein, a fluorophore, was discovered to provide the same effects as CALI using a continuous wave laser [3]. The subsequent discovery that incoherent light can produce similar effects further opened the possibility for high-throughput multiplex applications. These developments led to fluorophore-assisted laser inactivation (FALI), which is now the preferred choice. With fluorescein, the effective radius of ˚ , making it still within the range protein modification is 40 A of inhibiting specific protein domains [4,5]. Most applications of CALI/FALI have been based on a hypothesis-driven approach involving inactivating a known protein and trying to validate its role in a particular biological process or pathway. The application of FALI in a forward genetic-like screen was recently demonstrated [6]. In a functional proteomic screen of the extracellular proteome of a tumor cell for proteins involved in tumor cell invasion, FALI was used in combination with mass spectrometry and antibody technologies to reveal a novel essential role for extracellular hsp90a, a target for cancer therapy. The findings suggest new therapeutic approaches of inhibiting progression of cancer that may increase the efficacy and safety of the current generation of hsp90 inhibitors. Such discovery of 114
www.drugdiscoverytoday.com
Figure 1. Principle of chromophore-assisted laser inactivation (CALI). (a) A target protein is (b) complexed with a ligand (yellow; e.g. antibody, antibody fragments such as scFv and Fab, peptide, nucleic acid aptamers, small molecules) labeled with dye molecules (green; e.g. malachite green, fluorescein). The complex is irradiated with laser or incoherent light (red lightning) leading to (c) the generation of reactive species (concentric circles) that travel a short distance resulting in (d) modifications (black) of nearby amino acids. If the modified amino acids are responsible for a function, that particular functional protein domain will be inactivated, leaving the rest of the other functional domains intact and remain functional. In genetically targeted CALI or FALI, the chromophore is directly attached to the target protein. This can be accomplished using GFP genetically fused to the target protein, whereby GFP serves as the chromophore at the same time. Alternatively, a tetracysteine tag genetically fused to the target protein will specifically capture the chromophore (ReASH or FlASH) to facilitate CALI.
hsp90a’s extracellular location including its functional interaction with matrix metalloprotease 2 would have not been possible with genetic or RNAi approaches. This further highlights the importance of validating target function at the protein level (see Outstanding issues). Both CALI and FALI provide high spatial resolution inactivation of protein domains mimicking how a pharmacological agent will affect a target protein. However, one disadvantage with these methods is when the target protein turnover is high because of continuous synthesis of new proteins by the cellular machinery leading to a compensatory effect or negative CALI/FALI effect. For intracellular targets, the ligand should be delivered in sufficient quantities into the cell. Establishing controls to demonstrate that the ligand delivered into the cell is binding to the target protein intracellularly remains a challenge. Thus, negative results from CALI and FALI are not easily subject to interpretation.
Vol. 1, No. 2 2004
Genetically-targeted CALI Two recent approaches have addressed the possibility of efficiently targeting targets with CALI or FALI, circumventing the use of ligands specific to the target protein. At the same time, it addresses the limitations of CALI/FALI against intracellular targets. Instead of using a ligand that can be linked to a chromophore or fluorophore, a tag with specificity to chromophores or a fluorescent protein is directly fused to the target protein as a genetic fusion. Thus, the translated protein would have a high probability of being affected by CALI or FALI. In the first setting, a fluorescent protein (e.g. green fluorescent protein (GFP)) is genetically fused to a target protein. Rajfur et al. [7] used enhanced green fluorescent proteins (EGFP) for CALI towards understanding cytoskeletal structure by modulating the function of a-actinin through laser irradiation of EGFP-a-actinin expressed in Swiss 3T3 fibroblasts, leading to the detachment of stress fibers from focal adhesions (FAs). The CALI of EGFP–FAK (focal adhesion kinase) was spatially resolved in that only the kinase activity of FAK was inactivated, which is consistent with other experimental results. Although these studies have provided significant results, the efficiency of inactivation is not satisfactory compared to the use of organic dyes and may be limited by the fusion of a significantly large protein domain into the target protein that can severely affect protein function. In a second setting, to take advantage of the higher efficiency of organic dye molecules for generating reactive species instead of GFP for in vivo applications, a transgenically encoded tag was used that comprised a tetracysteine motif that specifically binds a modified form of fluorescein (FlAsH) and a membrane permeant, red biarsenical dye (ReAsH), calling the techniques FlAsH-FALI [8] and ReAsH-mediated CALI [9], respectively. FlAsH and ReAsH are only fluorescent upon binding to the tetracysteine motif. FlAsH-FALI and ReAsH-mediated CALI is specific and proportional to the duration of illumination and followed first-order kinetics. In an application of FlAsH-FALI, the authors engineered into synaptotagmin I (Syt I), a tetracysteine motif that binds the membrane-permeable, non-fluorescent fluorescein derivative 40 50 -bis (1,3,2-dithioarsolan-2yl) fluorescein (FlAsH) [8]. The engineered synaptotagmin I was introduced into Drosophila and was functionally expressed followed by the introduction of FlAsH to inactivate it. ReAsH-mediated CALI was successfully applied to inactivate connexin43 and a1C L-type calcium channels [9]. The authors further showed that ReAsH was much more efficient than FlAsH or GFP in generating reactive oxygen species leading to better inactivation. In addition to being more efficient than EGFP, the tetracysteine motif is only a few amino acids compared to GFP, making it more tolerable for protein fusions. However, the use of the teteracysteine motif might not be applicable
Drug Discovery Today: Technologies | Target validation
to proteins that are transported into the endoplasmic reticulum where the oxidizing environment might disrupt the motif. FlAsH-FALI and ReAsH-mediated CALI are analogous to the analog sensitive enzyme alleles (ASEA) methods described below but may be more versatile since it does not require prior knowledge or bias towards a functional site of the target molecule and the ligand does not have to be modified. Conceptually this elegant approach can be applied in a forward (see Glossary) genetic screen for tagging different genomics targets with the tetracysteine motif and randomly performing FlAsH-FALI and ReASH mediated CALI with the appropriate phenotypic screens.
Analog sensitive enzyme alleles The fundamental concept of ASEA is based on the modification of a ligand (e.g. small molecule) followed by the complementary modification of the receptor (e.g. target protein) facilitating specific interaction between the ligand and receptor [10]. The modified ligand is referred to as orthogonal in normal cells because it cannot interact with its receptor and preferably any other target protein in the cell. The new target receptor protein is engineered to accept the newly modified ligand. The newly engineered protein retains its normal physiological functions except for its new ability to interact with the modified ligand. More recent ASEA approaches have been applied to kinases, which regulate cellular processes and represent about 2% of all human genes. A set of analogsensitive kinase alleles (ASKA) and small molecule scaffolds were developed leading to an approach that can systematically probe the physiological roles of the different kinase families without the need for specific engineering of each small molecule and protein [11]. The key to ASKA is the ability to create a subtle yet distinct discriminant in one kinase, distinguishing it from the rest of the other kinases by modifying the ATP binding site. This mutation leads to ‘‘normal’’ functioning kinase with preference for a modified ATP analog. The amino acid residue targeted for mutation lies in the ATP binding site and is conserved among all kinases, making the modification of target kinases possible without knowledge of their three-dimensional structures. This approach has been successfully applied to a variety of kinases (reviewed in [12]). A most recent application that highlights the technology was demonstrated to a complex immunological system whereby novel p56 (LcK) ASKAs were used to show dose-dependent correlation of thymocyte development with varying Lck activity [13]. The ASEA principle has also been extended beyond kinases. A recent class of analogspecific enzyme alleles has been directed against protein methyltransferases [14]. The application of ASEA in vivo is a powerful demonstration of the technology’s potential. This was demonstrated in defining the role of myosin-1c (also known as myosin-1b) www.drugdiscoverytoday.com
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in the adaptation of sensory hair cells among the 40 different myosins implicated in the process [15]. ASEA circumvents the generation of multiple lines of mice expressing different levels of transgenes. ASEA will eventually be the method of choice in validating proteins that are amenable to the principle of orthogonal chemical genetics in vivo because it directly mimics how a pharmacological agent works. However, one limitation is that only functional domains that are amenable to orthogonal chemical genetics can be manipulated and other protein domains that are not amenable to analogs will be inaccessible.
Comparison of technologies With the plethora of targets one can take a ‘‘reverse’’ or hypothesis-driven approach whereby a putative target is modulated and its association with a particular biological process or pathway is deduced from a relevant phenotypic or biochemical assay. The limitation of this approach is that one has to sort individually through the thousands of different genes or proteins against multitudes of different assays, which can be tedious. In the ‘‘forward’’ approach, once a functional hit is identified, the corresponding target responsible for the modulation of the function or phenotype is deconvoluted. The advantage of this approach is that one
can sort through the proteome rapidly for the proteins relevant to a particular disease or biological pathway. One compromise is that certain targets might be missed out by virtue of the sensitivity and efficiency of the technology to modulate the particular target. From the cellular screen it is imperative that validation continues in a relevant in vivo model (see Outstanding issues). The aforementioned technologies (for summary see Table 1) are best suited for either the ‘‘reverse’’ or ‘‘forward’’ approaches. The ASEA technology would be very valuable for analyzing different families of proteins such as kinases in vivo. One disadvantage is that modulation is limited to the catalytic sites. It is known that proteins have additional functional domains that can be as good a target site as the catalytic domains. CALI/FALI for a large number of intracellular proteins can be slow, limited by the ability to deliver significant large number of ligands into the cells. However, the use of genetically targeted CALI/FALI offers a viable alternative. For large-scale cell-based screens it is reasonable to combine these genetically targeted CALI/FALI approaches with multiplex cloning to fuse the tetracysteine tags into different sites of the targets. Alternatively, one can have a non-biased way of inserting the tetracysteine tags into the targets by coupling with the CD tagging approach [16]. Although photodynamic
Table 1. Comparison summary table Name of specific type of technology
Chromophore/ flourophore-assisted laser/light inactivation (CALI/FALI)
Genetically targeted CALI/FALI
Analog specific enzyme alleles (ASEA)
Names of specific technologies with associated companies and company websites
Xstream and XCALIbur from Xerion Pharmaceuticals (http://www.xerion-pharma.com/)
GFP-CALI (Professor Kenneth Jacobson, University of North Carolina, http://www-cellbio.med.unc.edu/ jacobson/jacobso.htm) FlAsH-FALI (Professor Graeme Davis, University of California, San Francisco, http://www.ucsf.edu/davislab/)
P-inhibitor and P-target technologies from Cellular Genomics, Inc. (http://www.cellulargenomics.com/)
CALI and FALI (Dan Jay of Tufts University) (http://www.tufts.edu/ksloan01/ DanJay.html)
ASKA (Professor Kevan Shokat, University of California, San Francisco, http://www.ucsf.edu/shokat/)
ReAsH-CALI (Professor Roger Y. Tsien, UC San Diego, http://www.tsienlab.ucsd.edu) Reagents sold by Invitrogen (http://www.invitrogen.com/) Pros
- Acute and high spatial resolution - Amenable to extracellular targets
- Acute and high spatial resolution - Amenable to intracellular targets - In vivo applications
- Specific and acute - Directly mimics a small molecule agent - In vivo applications
Cons
- Intracellular targets are challenging
- Introduction of tags might disrupt protein function and not mimic the native state
- Limited to targets with amino acids amenable to modification - In vivo applications challenging
- Limited to targets with amino acids amenable to modification - In vivo applications limited to surface area that can be exposed to light
- Limited to proteins that have catalytic sites that are amenable to the orthogonal approach - Limited to catalytic sites and not other regulatory domains
Costs
Not available
Not available
Not available
References
[1–6]
[7–9]
[10–15]
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Related articles Hardy, L.W. and Peet, N.P. (2004) The multiple orthogonal tools approach to define molecular causation in the validation of druggable targets. Drug Discov. Today 9, 117–126 Ilag, L.L. (2003) Direct methods for modulating protein function. Curr. Opin. Drug Discov. Dev. 6, 262–268 Shokat, K. and Velleca, M. (2002) Novel chemical genetic approaches to the discovery of signal transduction inhibitors. Drug Discov. Today 7, 872–879 Ilag, L.L. et al. (2002) Emerging high-throughput drug target validation technologies. Drug Discov. Today 7, S136–S142 Henning, S.W. and Beste, G. (2002) Loss-of-function strategies in drug target validation. Curr. Drug Discov. 5, 17–21
Drug Discovery Today: Technologies | Target validation
its adoption and appreciation of the need to validate targets at the protein level (see Outstanding issues).
Outstanding issues Transition from nucleotide-based validation to protein-based validation. Application of protein-based validation in more in vivo models. Use of protein-based approaches in forward proteinetic screens. Accessibility to technologies and cost.
References
therapy has been shown to work, applications of CALI or genetically targeted CALI/FALI in relevant disease animal models in vivo remains to be seen. For the forward approach, the feasibility of using FALI in a functional proteome screen has been successfully demonstrated whereby randomly selected antibodies that gave a FALI effect were used subsequently for immunoprecipitation and the cognate target proteins identified via mass spectrometry [6]. Applications to more sophisticated screens based on use of phage display for selecting on cell surface proteins and other functional assays are anticipated to follow. Conceptually, genetically targeted CALI/FALI can be used in a forward screen. One can make random insertions of the tetracysteine tag into a cell or embryos followed by addition of FlAsH or ReAsH and subsequent illumination, then read out by a particular assay. Positive hits can then be recovered by identifying which gene has been genetically linked to the tetracysteine tag.
Conclusions The convenience of RNAi technology has led to a pause in the need to validate drug targets at the protein level in cell-based screens. However, once the genomic screens with RNAi are completed, the need for validation at the protein level will once more be appreciated. The emerging protein-based approaches should take advantage of this lull period to develop into more robust and cost-effective systems. In addition, use of these proteinetic methods in a forward approach such as proteome-wide functional screens will help increase
1 Jay, D.G. (1988) Selective destruction of protein function by chromophore-assisted laser inactivation. Proc. Natl. Acad. Sci. USA 85, 5454– 5458 2 Ilag, L.L. et al. (2000) Chromophore-assisted laser inactivation (CALI) to validate drug targets and pharmacogenomic markers. Drug Dev. Res. 49, 65–73 3 Surrey, T. et al. (1998) Chromophore-assisted light inactivation and selforganization of microtubules and motors. Proc. Natl. Acad. Sci. USA 95, 4293–4298 4 Beck, S. et al. (2002) Fluorophore-assisted light inactivation (FALI): a high-throughput tool for direct validation of proteins. Proteomics 2, 247– 255 5 Rubenwolf, S. et al. (2002) Functional proteomics using chromophoreassisted laser inactivation. Proteomics 2, 241–246 6 Eustace, B.K. et al. (2004) Functional proteomic screens reveal an essential extracellular role for hsp90 in cancer cell invasiveness. Nat. Cell Biol. 6, 507–514 7 Rajfur, Z. et al. (2002) Dissecting the link between stress fibers and focal adhesions by CALI with EGFP fusion proteins. Nat. Cell Biol. 4, 286–293 8 Marek, K.W. and Davis, G.W. (2002) Transgenically encoded protein photoinactivation (FlAsH-FALI): acute inactivation of synaptotagmin I. Neuron 36, 805–813 9 Tour, O. et al. (2003) Genetically targeted chromophore-assisted light inactivation. Nat. Biotechnol. 21, 1505–1508 10 Hwang, Y.W. and Miller, D.L. (1987) A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein. J. Biol. Chem. 262, 13081–13085 11 Bishop, A.C. et al. (2000) A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401 12 Shah, K. and Shokat, K.M. (2003) A chemical genetic approach for the identification of substrates of protein kinases. Methods Mol. Biol. 233, 253–271 13 Denzel, A. et al. (2003) Cutting Edge: a chemical genetic system for the analysis of kinases regulating T cell development. J. Immunol. 171, 519– 523 14 Lin, Q. et al. (2001) Design of allele-specific protein methyltransferase inhibitors. J. Am. Chem. Soc. 123, 11608–11613 15 Holt, J.R. (2002) A chemical-genetic strategy implicates myosin-1c in adaptation by hair cells. Cell 108, 371–381 16 Jarvik, J.W. et al. (2002) In vivo functional proteomics: mammalian genome annotation using CD-tagging. Biotechniques 33, 852–854
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