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REVIEWS 6 Wiegand, G. et al. (1998) Studies of structure and local wetting properties on heterogeneous, micropatterned solid surfaces by microinterferometry. J. Colloid Interface Sci. 196, 299–312 7 Heiduschka, P. et al. (1996) Microstructured peptide-functionalised surfaces by electrochemical polymerisation. Chem. Eur. J. 2, 667–672 8 Kalb, E. et al. (1992) Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers. Biochim. Biophys. Acta 1103, 307–316 9 Rädler, J. et al. (1995) On the phenomenology and kinetics of lipid bilayer spreading on hydrophilic surfaces. Langmuir 11, 4539–4548 10 Chen, C.S. et al. (1997) Geometric control of cell life and death. Science 276, 1425–1428 11 Naumann, C. et al. (1996) Hisactophilin-mediated binding of actin to lipid lamellae – a neutron reflectivity study of protein membrane coupling. Biophys. J. 71, 811–823 12 Lambacher, A. and Fromherz, P. (1996) Fluorescence interferencecontrast microscopy on oxidized silicon using a monomolecular dye layer. Appl. Phys. A 63, 207–216 13 Rothenhäusler, B. and Knoll, W. (1988) Surface-plasmon microscopy. J. Appl. Phys. 67, 3572–3575 14 Wegner, J. et al. (1996) Impedance analysis of epithelial and endothelial cell monolayers cultured on gold surfaces. J. Biochem. Biophys. Methods 32, 151–170 15 Albersdörfer, A. et al. (1998) High resolution imaging microellipsometry of soft surfaces at 3 mm lateral and 5 Å normal resolution. Appl. Phys. Lett. 72, 2930–2932 16 Kühner, M. et al. (1994) Lipid mono- and bilayer supported on polymer films: composite polymer lipid films on solid substrates. Biophys. J. 67, 217–226 17 Schmitt, L. et al. (1994) Synthesis and characterization of chelatorlipids for reversible immobilization of engineered proteins at selfassembled lipid interfaces. J. Am. Chem. Soc. 116, 8485–8491 18 Kloboucek, A. et al. Adhesion-induced receptor segregation and

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adhesion plaque formation: a model membrane study. Biophys. J. (in press) Berndt, P. et al. (1995) Self-assembling amphiphiles for construction of protein molecular architecture. J. Am. Chem. Soc. 117, 9515–9522 Kantlehner, M. et al. (1999) RGD-vermittelte Adhaesion von Osterblasten an Implantat-Oberflaechen. Angew. Chem. 111, 587–590 Löfås, S. and Johnsson, B.J. (1990) A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands. Chem. Soc. Chem. Commun. 1526–1528 Stelzle, M. et al. (1993) On the application of supported bilayers as receptive layers for biosensors with electrical detection. J. Phys. Chem. 97, 2974–2981 Hillebrandt, H. et al. (1999) High electric resistance thin polymer/ lipid composite films on indium-tin-oxide (ITO) electrodes. Langmuir 15, 8451–8459 Gritsch, S. et al. (1998) Impedance spectroscopy of porine and gramicidin pores reconstituted into supported lipid bilayers on indium-tin-oxide. Langmuir 14, 3118–1325 Baumgartner, P. et al. (1994) Fabrication of in-plane-gate transistor structures by focused laser beam-induced Zn doping of modulationdoped GaAs/AlGaAs quantum wells. Appl. Phys. Lett. 64, 592–594 Stelzle, M. et al. (1992) Two-dimensional microelectrophoresis in supported lipid bilayers. Biophys. J. 26, 1346–1354 McConnell, H.M. et al. (1992) The cytosensor Microphysiometer: biological applications of silicon technology. Science 257, 1906–1910 Fromherz, P. et al. (1991) A neuron–silicon junction: a retzius cell of the leech on an insulated-gate field-effect transistor. Science 252, 1290–1293 Loidl-Stahlhofer, A. et al. (1996) The thermodynamic control of protein binding to lipid bilayers for protein chromatography. Nat. Biotechnol. 14, 999–1002

Using photolabile ligands in drug discovery and development György Dormán and Glenn D. Prestwich Photoactivatable ligands are important tools used in drug discovery and drug development. These ligands enable researchers to identify the targets of drugs, to determine the affinity and selectivity of the drug–target interaction, and to identify the binding site on the target. Examples are presented from three fundamentally different approaches: (1) photoaffinity labeling of target macromolecules; (2) photoactivation and release of ‘caged’ ligands; and (3) photoimmobilization of ligands onto surfaces.

hotoprobes are becoming increasingly common as remotely controllable tools in the drug-discovery process1–6 (Fig. 1). One can take advantage of the dark stability and light sensitivity of photoprobes in two stages. First, the photoreactive group is introduced chemically into a biologically active molecule and the parameters for the biological tests are established. Second, the specific photochemical reaction is

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G. Dormán ([email protected]) is at ComGenex, Budapest, 1027 Bem rkp. 33-34, Hungary. G.D. Prestwich (gprestwich@deans. pharm.utah.edu) is at The University of Utah, Department of Medicinal Chemistry, Salt Lake City, UT 84112-5820, USA.

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then initiated by irradiation of the photophore at a defined wavelength, thereby either forming a new covalent linkage (photoaffinity labeling and photoimmobilization) or cleaving a specific bond (photodeprotection). The increasing miniaturization of assays and the use of high-throughput screening (HTS; see Glossary) techniques now widely exploit these photochemical tools through immobilization of small molecules, biopolymers and spatially addressable combinatorial libraries7,8. In this review, we present examples, primarily from the past four years, to illustrate how these techniques can contribute to the drug-discovery process. In addition, we note how photoactivation techniques have

0167-7799/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(99)01402-X

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Glossary Assembly protein 2 Azide Brominated 7-hydroxycoumarinyl methyl esters BP Benzophenone Bpa 4-Benzoyl-phenylalanine BZDC-IP3 4-Benzoyldihydrocinnamoyl-P-1-Oaminopropyl Ins(1,4,5)P3 DIA Diazirine HTS High-throughput screening Ins(1,4,5)P3 Inositol 1,4,5-triphosphate MALDI-TOF MS Matrix-assisted laser-desorption and ionization coupled with time-of-flight mass spectrometry MDR Multidrug resistance MTP Microsomal triglyceride transfer protein NSAID Non-steroid anti-inflammatory drug P-GP P-glycoprotein PAL Photoaffinity labeling PE Phycoerythrine PKA Protein kinase A PKI Protein kinase inhibitor PtdIns(4,5)P2 Phosphatidyl inositol-4,5-bisphosphate PtdIns(3,4,5)P3 Phosphatidyl inositol-3,4,5-trisphosphate SPR Surface plasmon resonance uPA Urokinase-type plasminogen activator

Early lead

AP-2 AZ Bhc

Target identification

Photoaffinity labeling

Compound libraries for screening

High-throughput screening

Photocleavable linkers for solid-phase synthesis

Photoimmobilization of biopolymers

New Lead

generated new ideas and opportunities for the development of novel, innovative methodologies. In particular, we have selected examples that show how photolabile ligands can be used to answer five major questions. • What is the ligand for this target protein? • What is the protein target for this ligand? • What are the affinity and selectivity for this ligand? • Where and when are the target proteins found? • What is the active site of this target? General principles of the main photochemical techniques Two main classes of photochemistry can be distinguished, which depend on whether or not a chemical bond is formed or cleaved (Fig. 2). In photoaffinity labeling (PAL), a new covalent linkage is created between a light-sensitive, detectable ligand and a biopolymer upon irradiation, in a reversibly bound state. As a result of the photoinitiated coupling reaction, the ligandbinding site of the biopolymer (e.g. a receptor protein or an enzyme) becomes irreversibly occupied, and can inactivate or activate the biological function of the biopolymer. The photoligand must contain a readily detectable tag (e.g. radioactive, fluorescent or immunoreactive), which allows ready determination of the macromolecule and, in particular, the fragments of the molecule that have been covalently modified1. A similar photochemical process is used for photoimmobilization of biopolymers or ligands to create modified surfaces or patterned arrays (Figs 3 and 4). In the inverse process, the key action is a photoinduced release of bioactive compounds from a ‘caged’ ligand6,9. The ‘caging’ group is used to mask the biologically active pharmacophore; the caged ligand should show no biological activity in the dark. Often, the caging group also alters the cellular uptake or distribution of the caged TIBTECH FEBRUARY 2000 (Vol. 18)

Binding-site model

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trends in Biotechnology

Figure 1 Photochemical tools used in the drug-discovery process.

ligand in an experimentally desirable fashion. Irradiation causes the bond between the ligand and the photosensitive group to be broken. This technique enables the investigation of fast kinetic processes (in the submillisecond range) including neurotransmitter release and cell signaling through second messenger pathways. Photochemical triggering is also an important method for initiating specific structural or biomolecular changes in situ. Photoaffinity labeling PAL was developed over 30 years ago, but recently a new era of applications has appeared as a result of the development of more-efficient photophores, new highresolution separation methods and detection techniques with higher sensitivity3,10,11. The photocovalent

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Figure 2 The main principles of photoaffinity labeling and photolytic release from caged compounds. (a) Photoaffinity labeling (photocovalent attachment); dotted line represents a new covalent linkage. (b) Photorelease of bioactive ligands from caged compounds; dotted line represents a photocleavable bond.

modification of a macromolecule can provide valuable information about the location and architecture of the ligand-binding site. Identification of drug–target interactions at three levels The starting point of the modern drug-development process is the identification of specific targets associated with a particular disease state; knowing the target and its function enables the creation of molecules that can selectively manipulate biological processes. These target proteins are often differentially expressed in tissues, which further facilitates the development of a selective therapy. The emergence of proteomics has accelerated the target-hunting process in different tissues and organs exhibiting a specific pathology12. Bioactive ligands should show selective recognition to achieve a specific response to restore the healthy state. However, therapeutic molecules might have multiple or secondary binding sites in living systems, which

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can be responsible for side effects or toxicity. Drug metabolism and binding to carrier proteins are events that can be approached with a photolabile drug analog. PAL provides a direct and general method to identify binding proteins and also enables the possibility of direct identification of the interacting site at the molecular level. Therefore, PAL can provide three levels of identification in a single experiment. Macro level First, PAL enables the identification of primary binding proteins, one of which should mediate the pathology of interest (on the macro level). Such information is often sufficient for biomolecular screening, which examines either individual drug candidates or, recently, diverse molecular libraries that have been synthesized by combinatorial organic synthesis. The information on secondary binding sites helps design drugs with better bioavailability, less toxicity or less cell-specific resistance. A simple PAL experiment carried out in the presence or absence of competitors with known biological effects might offer an alternative to in vitro binding assays; such an application can be extended to HTS (Ref. 13). Semi-micro level Second, a tagged protein-target isolated on the macro level can be fragmented into short peptides to localize the binding region within the biopolymer (semi-micro level). This stage enables mechanistic and comparative studies on the binding interaction. Using these data, recombinant binding domains can be expressed, purified and used in cellular or biophysical studies to further explore the target–ligand interaction. Micro level Third, the three-dimensional structure of the ligand– biopolymer complex, particularly the conformation of the binding pocket with key amino acids involved in the binding interactions, provides the most valuable information for structure-based drug design (micro level). In principle, X-ray crystallography and solution phase high-resolution NMR would be the best methods to achieve this level of information. These powerful analytical techniques are supported by recombinant DNA technology, which can produce milligram amounts of polypeptides or polynucleotides. There are currently a variety of cases in which structural analysis at the micro level is unachievable or difficult to accomplish, such as in transcription factor complexes, ribosomes, vesicular-coat proteins, local cytoskeletal structures and membrane-embedded signaling complexes. In such cases, PAL provides an important method for subunit identification and active-site mapping, which complements site-directed mutagenesis and antibody-recognition studies. Outline of the PAL experiments The design of the photoprobe is greatly enhanced by an extensive structure–activity relationship study. Based on the available data, a photophore is appended or introduced and the bioactivity of the new analog is reexamined. Ideally, the photoprobe should be bioactive in the same range as its parent compound, but compounds with as much as 1000-times lower activity can still be useful. The next step is to synthesize the bioacTIBTECH FEBRUARY 2000 (Vol. 18)

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Figure 3 Two different approaches for the photoimmobilization of biopolymers: (a) shows the photoattachment of a protein-photophore adduct to a surface; (b) shows prior chemical modification of the surface followed by photoattachment in order to immobilize a macromolecule.

tive photoprobe in a radiolabeled form. The original synthesis of the unlabeled molecule is best developed with this eventuality in mind; alternatively, nonradioactive labels (primarily biotin) can also be attached via a linker arm14,15. In the photoaffinity experiment, a tissue homogenate is prepared and the detergent-solubilized membrane pellet and the supernatant are then both examined. In addition, our experience indicates that unless an initial protein purification step is used, potentially useful photoprobes might be missed because of high nonspecific background labeling. Step-gradient ion exchange, hydrophobic or affinity (e.g. heparin-agarose) separations can give relatively few fractions, each of which can be analysed by PAL (Ref. 16). Fractions are incubated with the photoprobe to establish a noncovalent interaction before irradiation. Irradiation at the excitation wavelength establishes a covalent linkage between the ligand and an appropriate target protein. Labeled protein mixtures are separated by denaturing-gel electrophoresis; labeled bands are detected with a phosphorimager or, for weak b-emitters such as tritium, by fluorography on X-ray film. After a labeled protein has been found, competitors are co-incubated with the photoaffinity label and the target protein to establish whether or not the PAL is selective. The specifically labeled proteins are distinguished as those that are competitively displaced by a 100–1000-fold excess of the parent ligand. TIBTECH FEBRUARY 2000 (Vol. 18)

The specifically labeled proteins are isolated and subjected to limited proteolytic digestion or chemically induced cleavage at specific residues. The most-radioactive fragments are localized by electrophoretic or HPLC separation, and the size and composition can be analysed by matrix-assisted laser-desorption and ionization, coupled with time-of-flight (MALDI–TOF) mass spectroscopy or by Edman degradation. Requirements of the photoprobe There are a limited number of generally useful photoreactive groups used in PAL. The photoreactive groups should have: (1) reasonable stability under ambient light; (2) a photochemically generated excited state with a lifetime shorter than the dissociation of the ligand–receptor complex but long enough to spend sufficient time in close proximity to a target site for covalent linkage; (3) an unambiguous photochemistry to provide a single covalent adduct; and (4) an activated form that reacts with C–H groups as well as nucleophilic X–H bonds. The last criterion is important in order to obtain a stable covalent linkage that survives micro level analytical methods and avoids the diffusion or release of an activated form that results in solvent trapping and nonspecific labeling. The activation wavelength of the photophore should be longer than the ultraviolet absorption for most proteins

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Photolithography: spatially addressable combinatorial chemistry

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Figure 4 Application of photocleavable groups in spatially addressable, photolithographic synthesis of combinatorial libraries. A, B, C....-Pr represents protected amino acids or nucleotides.

(.300 nm). Several comparative studies11,17 have shown that reliable and reproducible high-efficiency labeling of target proteins is obtained using tetrafluorophenyl azides (AZ), trifluoromethylphenyl diazirines (DIA) and benzophenone (BP) photophores (Fig. 5). In our experience, the BP photophore performs the best; it can be activated in a reversible manner via excitation–relaxation cycles, and only C–H bonds within 3.1 Å of the carbonyl oxygen are modified. Moreover, BPs are stable to common protic solvents, react only with target proteins and generally lead to highly efficient single-site photocovalent modification5,10. Specific applications of PAL Several new regulatory mechanisms have been discovered in recent years, including endogenous ligands with their receptors, and substrates with their converting enzymes18. The precise target of action remains to be identified for many potent drugs that are in development, or even in use. These two trends have led to an increased use of PAL, as reflected by the broad variety of applications in the literature. Potential drug targets Plasminogen activation by a receptor-bound urokinase type activator (uPA) is important in tumor metastasis.

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Identification of an interaction between a uPA and its receptor would provide a new target for the development of non-peptidic antagonists. These data and the photoaffinity analog of a photoreactive decapeptide antagonist labeled Arg53 and Leu66 led to a proposed molecular topology for the receptor–ligand interface. This model was then used as a starting point for the design of small-molecule antagonists19. Selectivity between specific ligand families is sometimes difficult to identify clearly. For example, the angiotensin family consists of an octapeptide and its truncated analogs, whose physiological functions in the central nervous system are difficult to separate; the hexapeptide AngIV might be involved in memory acquisition through a receptor. Using photolabile analogs of AT4 (Figs 6a,b), an integral membrane glycoprotein can be specifically labeled (186 kDa) in bovine aortic endothelium. These peptide analogs provide effective photoprobes to identify AT4 receptors in different tissues20. In another example, formyl peptide receptors induce chemotaxis and defense processes, which produce inflammatory mediators. The bindingsite topology of a formyl Met-Leu-Phe tripeptide target was determined by a fluorescently labeled formyl peptide containing photolabile 4-benzoyl-phenylalanine (Bpa). Upon irradiation, this analog labeled a transmembrane domain between the 70 and 90 amino acids of the receptor from chinese hamster ovary cells21. In addition, PAL showed that a pituitary growth-hormone-releasing peptide receptor subtype (57 kDa) was different from one that had been previously cloned22. In a last example, cholecystokinin-A receptor interactions were directly probed with photolabile 1,5-benzodiazepine agonists and antagonists23. Rühmann et al.24 investigated the 41-residue polypeptide corticotropin-releasing factor, which is believed to synchronize the endocrine response to stress. The photolabile DIA-containing peptide analog specifically labeled a 75 kDa glycosylated protein in rats; such a receptor would be a potential target to reduce stressrelated symptoms24. Parathyroid hormone (PTH) regulates the blood plasma calcium level and bone remodeling; its action is exerted via a specific G-proteincoupled-binding protein (PTH1-Rc). Using PAL, Bisello et al.25 investigated this binding interface by successively replacing the N-terminal amino acids with Bpa; this photoaffinity-scanning approach led to the identification of Met414 and Met425 within transmembrane helix 6, as a putative contact domain. Typically, the primary goal of applying PAL (Ref. 18) is the identification of putative molecular targets of certain drug or drug-candidate molecules. The binding site of the sulfonyl urea diabetes drug, glibenclamide, was recently identified in insulin-secreting cells, by PAL, as a 38 kDa protein tightly coupled with a 160 kDa polypeptide26. A novel anti-inflammatory and analgesic drug, a-trinositol, is a noncompetitive antagonist of neuropeptide-Y. PAL experiments using a photolabile analog revealed specific binding proteins in different tissues (human blood platelets and umbilical cords) as 55 kDa and 43 kDa polypeptides27, suggesting that these uncharacterized proteins might mediate the activity of this drug candidate. Also, potential targets of the anti-fungal semi-synthetic cyclic lipopeptide, echinocandin, have been identified in TIBTECH FEBRUARY 2000 (Vol. 18)

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Figure 5 Photochemical events of three major photophores used in photoaffinity labeling: benzophenone, acrylazide and diazirine. (a) Benzophenone photochemistry, (b) aryl azide photochemistry and (c) diazirine photochemistry.

Candida albicans by PAL, revealing an unknown 40 kDa protein28. PAL can also be used to identify either the binding sites of catalytic domains or the substrates of enzymes. First, NO-synthase oxidizes L-arginine to L-citrulline and NO with the aid of a cofactor, tetrahydro-L-biopterin. BP-linked photolabile antagonists were designed to identify the pterin binding site and subsequent PAL experiments have identified a 341-amino-acid sequence in the oxygenase-dimerization domain of NO-synthase29. A new family of cholesterol-lowering agents has an inherent photolabile BP unit in the pharmacophore. One of these, Ro48-8071 (Fig. 6c) is a potent, noncompetitive inhibitor of oxidosqualene cyclase. During irradiation, the enzyme is specifically modified by the TIBTECH FEBRUARY 2000 (Vol. 18)

tritium-labeled drug candidate and competition experiments have revealed that it shares a common binding site with substrate-like inhibitors30. Subsequently, the drug-binding site has been established and a molecular model suggests that the drug blocks the entry of substrate to the catalytic site31. The steroid 5a-reductase (5a-R) produces 5adihydrotestosterone; elevated levels of this potent androgen cause prostate hyperplasia and can lead to prostate cancer. Therefore, this membrane-bound enzyme is an attractive target for the therapy of prostate disorders. A photolabile, BP-containing steroid analog specifically incorporated into a 26 kDa protein causes a complete loss of catalytic activity32. In a similar fashion, a PAL inhibitor has been developed to study the subsequent

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Figure 6 Photoaffinity probes used in the identification and characterization of selected drug targets. Applications for compounds (a–h) are described in the text.

events in the microsomal lipid-transfer pathway, which is important for secretion of apoB-lipoproteins. Irradiation of the microsomal triglyceride-transfer protein (MTP) and a BP-containing photoaffinity inhibitor (BMS 192951) blocks the complete pathway, confirming that MTP plays a key role in lipid transfer33. Cyclooxygenase-1 is the common molecular target for non-steroid anti-inflammatory drugs (NSAIDs), such as aspirin and ibuprofen. It is an integral membrane protein, but lacks transmembrane domains; the topology of the membrane-associated region, which is an amphipathic helix, was postulated to contain the catalytic site. The region was identified by a nonpolar DIA probe (Fig. 6d), which caused broad labeling on the protein region within the membrane; the labeling was competitively diminished by the standard NSAIDs (Ref. 34). Signal transduction Within the cell, second messengers change rapidly with time and space in response to surface-receptor

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activation. Both production and degradation of second messengers are under tight enzymatic control. Selectivity is encoded by receptor affinity, receptor localization and allosteric sites. Defects or alterations in the signaling pathways often lead to inappropriate up- or down-regulation of second messengers, leading to pathologies commonly observed in cancer. The discovery of the complete transductory machinery and its control points is important for the development of specific drugs that can restore normal cell signaling. PAL techniques help to identify multiple binding sites for a specific ligand and to determine the binding specificity between the ligands. A photoactivatable analog of inositol 1,4,5-triphosphateIns(1,4,5)P3 [Ins(1,4,5)P3] was prepared by connecting a BPphotophore containing heterobifunctional crosslinker reagent to an amino-alkyl-tethered inositol polyphosphate analog (Fig. 6e). The photoaffinity labeling of the Ins(1,4,5)P3 receptor35 has been followed by several more-recent applications. For example, the TIBTECH FEBRUARY 2000 (Vol. 18)

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Figure 7 Photochemical transformations of four major photocleavable groups used in caged compounds. (a) o-Nitrobenzyl photochemistry, (b) benzoin photochemistry, (c) o-cinnamoyl photochemistry and (d) m-nitrophenyl photochemistry.

molecular similarity of the 4-benzoyldihydrocinnamoylP-1-O-aminopropyl Ins(1,4,5)P3 (BZDC-IP3) to phosphatidyl inositol-4,5-bisphosphate [PtdIns (4,5)P2] has resulted in the elucidation of two binding sites. Human profilin (14 kDa) acts as a cellular regulator of the remodeling of the actin cytoskeleton. Successful photolabeling using BZDC-IP3 has enabled the identification of Ala1 in the N-terminal helix as the hydrophobic contact point for the BP photophore. Molecular modeling studies considering the attachment point (Ala1) and the maximum span of the linker revealed that two adjacent arginines (Arg135 and Arg136) at the end of the C-terminal helix, interacted electrostatically with the 4,5-bisphosphate moiety36. In another example, BZDC-IP3 has been used successfully as a photolabile mimic in the identification of PtdIns(4,5)P2, labeling the known pleckstrin homology domain of the phospholipase C d1 isozyme; thus PAL has revealed a substrate-docking site distinct from the catalytic site37. Transport processes A complete panel of photoaffinity analogs of inositol and phosphoinositide polyphosphates (InsPns and TIBTECH FEBRUARY 2000 (Vol. 18)

PtdInsPns) are available to examine the binding-specificity between different targets that are important in cytoskeletal remodeling, signal transduction, endo- and exocytosis and vesicular trafficking38,39.. One of the major components of the clathrin-coated vesicle is assembly-protein-2 (AP-2), which has been labeled with several photolabile InsPn analogs. Using an InsP6 photoprobe, competition experiments show ligand affinity in the order: InsP6.PtdIns(3,4,5)P3 .Ins(1,4,5)P3 .Ins(1,3,4,5)P4 .PtdIns(4,5)P2 .ATP (Ref. 40). In the non-clathrin pathway, Golgi coatomer is a heteroheptameric complex in coated vesicles that mediates traffic between the endoplasmic reticulum and the Golgi. Among the PtdInsPn photoprobes, BZDCIns(1,3,4,5)P4, a crude mimic of PtdIns(3,4,5)P3, selectively labeled the a-subunit, not previously known to mediate PtdInsPn interactions. Labeling and displacement studies using BZDC-PtdInsPn triesters suggested that both D-3 and D-5 phosphates were critical for recognition41. The coatomer study underlines the uniqueness of PAL for obtaining this result. Because efforts to dissociate the coatomer complex abrogated binding, it was necessary to photoaffinity label the intact heptameric complex; the subunit bearing the

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label can be determined after subunit dissociation and separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Therapeutically important transport processes have been extensively examined using PAL, for example, multidrug resistance (MDR)42. P-Glycoprotein (P-GP) is a drug-efflux pump, which has a binding affinity for structurally unrelated antitumor agents. This 1280amino-acid, 12-transmembrane-domain membrane protein is responsible for MDR, a major obstacle in cancer therapy. Several photoaffinity probes have been developed to reveal the major structural elements involved in the binding, enabling MDR reversing agents (chemosensitizers) to be designed. Using a BPcontaining pharmacophore43, Ca21-channel blockers, such as the dihydropyridines (chemosensitizers), were found to share the binding site with antitumor agents only partially. In a separate project, the binding site of the antimitotic agent, paclitaxel, was mapped by two different BP-containing analogs introduced at the 39and 7-positions44. The labeled peptide fragments were identified with specific antibodies that indicate that the 39-BP analog (Fig. 6f ) is covalently attached to a 103residue peptide (985–1088), whereas the 7-BP derivative (Fig. 6g) is bound to a 77-residue polypeptide (683–760) in one of the extracellular loops. Because the BP analogs can be excited at 360 nm, the irradiation does not damage P-GP, unlike analogs containing azidophenyl photophores45 (Fig. 6h). Photoimmobilization Photoimmobilization uses the same principle as PAL in the sense of photocovalent attachment, and therefore the same photophores (AZ, DIA, BP) can be used to covalently modify surfaces and solid supports7. The photophores are connected to a small ligand with a spacer to maintain full biological activity of the pharmacophore after immobilization. Two different approaches can be used. The first approach can be used in several different ways (Fig. 4). (1) An anticoagulant sulfated hyaluronic acid polymer can be immobilized on poly(ethylene terephtalate) by pattern-specific photocovalent modification with azidophenyl groups46. Platelet adhesion is significantly reduced around the micropatterns indicating that bioactivity is preserved during immobilization. (2) The Arg-Gly-Asp (RGD) sequence in polypeptides that induces cell adhesion can be studied using photolithographic micropatterning. BP-containing RGD analogs are allowed to form self-assembled monolayers and are then photoimmobilized47. The pattern is visualized by autoradiography when the radiolabeled ligand is attached. (3) Photoactivatable glycoaryldiazirines are used for mask-assisted photolithographic surface glycoengineering48. (4) Microscale gradients of proteins are generated using immobilized ligands (phycoerythrine, PE) to study cell migration. The BP-tetraethylene glycol-PE conjugate is photolytically attached to polystyrene surfaces and biological responses are examined in a biopolymer gradient49. In the second approach, for example (Fig. 3), BP photophores are attached to a silica surface and used as an immobilized triplet sensitizer50. Following that principle, solubilized proteins and other biopolymers can be linked to the surface upon irradiation. The BP-derived

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diradicaloid induced H-abstraction and random, multipoint photocoupling to the surface; the protein retained most of its biological activity. This technique can be used for HTS. Caged compounds Basic concepts Photochemical activation of biomaterials have recently been classified51 as either: ‘single-cycle’ photoswitches, in which irradiation of photocleavable functional groups causes instantaneous activation via a bond cleavage; or ‘multicycle’ photobiological switches, wherein the biologically active and inactive states can be accessed by irradiation with different wavelengths, providing reversible photochemical control. For single-cycle photoswitches, speed is the critical factor and is achieved by laser photolysis of photocleavable protecting groups, such as o-nitrobenzyl, desyl, trans-o-cinnamoyl and m-nitrophenyl photophores (Fig. 7). These photophores enable the rapid photorelease of bioactive materials from inactive caged precursors9. As ligand–protein interactions occur on a submillisecond timescale, the rapid and complete delivery of bioactive compounds is essential to study the fast kinetic events (either as a change in the concentration of chemical messengers and ions or as conformational changes) triggered by ligand binding. In addition, this technique allows spatial control within a cell and therefore provides a powerful alternative to microinjection. The design of photoreleasable ligand precursors starts with structure–activity relationship data similar to that used for PAL. However, in this case the primary goal is to mask the key elements in the pharmacophore to produce a compound that is ‘dark inactive’ before photolytic activation. Many caged compounds suffer from incomplete ‘dark inactivity’, which complicates the interpretation of experimental results. Examples include caged neurotransmitters, phosphoinositides and other signaling molecules. Caged neurotransmitters, phosphoinositides and other signaling molecules Caged neurotransmitters can provide unique and valuable information about fast synaptic responses in the central nervous system. This tightly regulated synaptic transmission uses excitatory and inhibitory amino acids, and their receptors are involved in learning, memory, neurological disorders (e.g. epilepsy) and anxiety. Thus, the elucidation of the activation mechanism is important for the design of new therapeutic agents. Niu et al.52 reported doubly-caged kainate analogs (Fig. 8a), which are activated by laser-flash-photolysis at 333 nm. The liberated neurotransmitter opens the cation channel and induces whole-cell current within ,1 ms. The non-NMDA glutamate-receptor family, which includes kainate receptors, also mediates fast excitatory synaptic transmission. Rossi et al.53 developed an efficient caged glutamate, which has low ‘dark activity’ and releases glutamate within the millisecond timescale in a pH-dependent manner. Alternatively, photoreleasable desyl-protected GABA and glutamate (Fig. 8b) analogs have been described by Gee et al.54; flash photolysis releases the active neurotransmitters within nanoseconds. To mimic enzymatic release, a TIBTECH FEBRUARY 2000 (Vol. 18)

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(a)

O

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trends in Biotechnology

Figure 8 (a–h) Using photocleavable groups: caged compounds, photoprodrugs, photocleavable linkers for solid-phase synthesis and a combined photoaffinity-photocleavable reagent.

photolabile cholinesterase inhibitor has been developed to photolytically release choline55. An important new innovation in neurotransmitter release is the development of two-photon caging groups. Furuta et al.56 pioneered this approach using the brominated 7-hydroxycoumarinyl-methyl esters (Bhc) and carbamates attached to glutamate. Bhc-glu requires less energy for activation and gives more-efficient localized uncaging. Ca21 release from intracellular stores is one of the major events in signal transduction. The time course TIBTECH FEBRUARY 2000 (Vol. 18)

and spatial localization of this process can be studied using caged Ins(1,4,5)P3 (Ref. 57). Many related photoactivatable inositol polyphosphates have been prepared in cell-permeant form58 and used for cellular studies. Caged, cell-permeant PtdIns(3,4,5)P3 is synthesized and used as a signal in cells to simulate phosphoinositide 3-kinase activation using the ligand alone59. Sphingosine phosphates modulate cell growth using Ins(1,4,5)P3-independent signaling via Ca21-ion mobilization. Qiao et al.60 developed caged analogs to

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study the pathways involved in the process and found that photolysis of the inactive precursors results in an increased level of cell proliferation. A ganglioside photoaffinity label has been used to localize the GD1bbinding site on tetanus toxin C, which was determined by MALDI-MS to be a single residue, His1293 (Ref. 61). A photolabile analog (Fig. 8c) of the apoptosisinducer L-leucyl-L-leucine methyl ester exhibits cytotoxic activity against natural killer cells of the human immune system62. For immunological studies, two caging groups were examined: o-nitrobenzyl and transo-cinnamoyl. After photolysis, both caged analogs release the active ligand and cause apoptosis. Caged polypeptide probes Recently, the ‘caging’ technique has been extended to photolytic deprotection of polypeptides in vitro and in vivo63. Kinase-mediated protein phosphorylation is an important element in the signal transduction of cell growth and proliferation. The identification of individual proteins in the signaling cascade can provide new drug targets for anticancer treatment. A site-directed photolabile analog of a proteinkinase-inhibitor peptide (PKI) has been developed that masks Arg5, which is responsible for the inhibitory activity. After photolysis, the inhibitory activity is completely recovered64. Wood et al.64 also developed a creative combination of active-site labeling and photorelease. The key step in the activation of protein kinase A (PKA) is a cAMP-dependent release of the catalytic subunit. From previous studies using PKI affinity labels, the catalytic site of the PKA is known to be near Cys199. Because other cysteine residues are also present in PKA, site-directed introduction of the caging group has been accomplished by attaching it to a short inhibitory peptide, thus targeting the caging reagent specifically to Cys199 (Ref. 65; Fig. 8d). The masked catalytic activity is readily recovered by irradiation. A post-synthetic modification of Cys199 is also achieved by carefully controlling temperature and pH conditions66,67. A biosynthetic approach to site-specific incorporation of unnatural amino acids, such as o-nitrobenzylprotected serine, has been developed68. In a model experiment, the photolabile amino acid was incorporated into peptide sequences of a thermostable DNA polymerase. A serine residue was protected between ‘exteins’ and ‘inteins,’ which are analogous to exons and introns in nucleic acid sequences. During photoinduced self-splicing, excision of the intein and ligation of the N-terminal and C-terminal exteins produces the functional protein. A tRNA misacylation approach has been used for protein engineering to prepare HIV-1 protease containing a caged asparate69. Similarly, an o-hydroxycinnamate ester of the serine in thrombin, at the cleavage site of the intramolecular serine protease activity, has been synthesized. An irreversible inhibitor that covalently modifies specific serine residues delivers the photophore. Photolysis at 366 nm produces thrombin activity, leading to coagulation of platelets. In the eye, abnormal blood vessels cause blindness: thus, the use of light-induced blood clotting might provide a novel method for the elimination of neovascularization70.

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Photoprodrugs Other in vivo applications include photodynamic therapy, which is currently under development for cancer treatment. The DNA-alkylating site of these prodrugs is blocked by a photocleavable group to give non-cytotoxic prodrugs. Local irradiation releases the cytotoxic agent and inhibits cell growth. For example, caged 5-fluoro-deoxyuridine (Fig. 8e) releases the anticancer agent upon irradiation at 350 nm (Ref. 71). In another application, phosphoramide mustards are masked by an o-nitrophenyl group to give inactive prodrugs (Fig. 8f ). These photoalkylating agents are successfully uncaged by light at 340 nm (Ref. 72). Two novel approaches to photodynamic cancer chemotherapy include the use of polymer-conjugated photosensitizers and labile cobalt–carbon bonds. (1) The polymeric derivatives exhibit increased uptake and retention by tumor cells, thus concentrating the photosensitizer73. (2) An infrared-sensitive Co-alkyl bond on a vitamin B12 derivative of chlorambucil can be used for targeted uptake followed by selective, local photolytic release of the active drug74. Photocleavable linkers and cross-linkers The photocleavable group can be placed in the middle of a side-chain or linker-arm. Heterobifunctional cross-linker reagents can tightly connect peptide subunits, peptide-antibody conjugates and peptide-fluorophore linkages. Ottl et al.75 reported a photocleavable cross-linker reagent with thiol- and amine-specific reactive functionalities connected to the o-nitrobenzyl photophore. The thiol-selective site is linked to thiolated dextran; the other end of the photophore is connected to lysine residues in G-actin. The dextran masks the active site of G-actin. After photolysis at physiological pH, full activity of the G-actin is recovered and rapidly polymerized to F-actin filaments75. Photocleavable biotin-containing heterobifunctional reagents that make the detection and isolation of biomolecules easier, and allow disconnection of the reporter group following isolation have also been developed76. The same principle has been applied to the photocleavable linkers in solid-phase combinatorial synthesis. Several modified o-nitrobenzyl-based linkers (Fig. 8g) were tested77 and after completing several combinatorial cycles of reaction, the library could be disconnected from the solid support by irradiation at 360 nm. Spatially addressable combinatorial synthesis of peptide and DNA libraries has been developed8. The key feature of this parallel synthesis technique is the combination of photolabile protecting groups and lithography (Fig. 4). It allows a pattern-directed photolytic cleavage in each cycle, followed by a coupling reaction with a new amino acid or a nucleotide, protected, again, with photolabile groups (Fig. 8). The sequence diversity is generated by the different patterns in each cycle. This principle has been used to achieve spatially defined immobilization of biomolecules with caged biotin. In a model experiment, caged biotin linked to serum albumin was immobilized on a glass surface. A photomask was applied in the presence of avidin, upon irradiation a specific pattern was created on the basis of the interaction between avidin and photoreleased biotin at the uncovered sites76. TIBTECH FEBRUARY 2000 (Vol. 18)

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Phototriggered DNA cleavage is a major tool used for studying the conformational changes and strand breaks, as well as for studying activation of nucleic-acidtargeted drugs, such as antisense oligonucleotides. The photolabile group is incorporated into a DNA sequence between two phosphates, and laser-pulse photolysis cleaves the bridge creating two short oligonucleotides78. In a related approach, photoinduced one-electron oxidation of DNA initiates a sitespecific cleavage and identifies the sites in a sequence that are most susceptible to strand scission. The application of a p-cyano-substituted BP derivative as an intercalating triplet sensitizer gives a supplementary feature to that photophore used generally in photoaffinity labeling79. A novel combination of PAL and a photocleavable linker was reported80 in a study of arthropod-derived neurotoxins. Ligand–receptor interactions were studied using the standard PAL technique forming a covalent linkage between them. The first photolysis was conducted under neutral conditions (Fig. 8h). The ligand was tagged with biotin as a detection device; binding to avidin beads facilitated isolation of the adduct. To recover the unlabeled ligand, a second photolysis was conducted under basic conditions, which severed the ligand–biotin connection. The purified peptide was finally sequenced by the multistage mass spectroscopic technique80. A related technique has been developed independently and used for identifying the active site of a glycosyltransferase15. Future perspectives The discovery of new therapeutic agents requires a coordinated interdisciplinary effort. Emerging techniques are accelerating the process from molecular biology and genomics to combinatorial synthesis and HTS. At each step, photochemistry provides mild, controllable and selective tools for scientists engaged in the drug-discovery process. In the future, additional innovative applications can be anticipated to increase the efficiency and resolution of the photochemical methods. Combining photolabeling, photoreleasing, photoimmobilization and the increased use of two-photon approaches and multistage switches constitutes areas for important future developments in drug discovery. The possible use of fluorescence resonance energy transfer to excite a photophore could provide a new approach to HTS. In addition, caged compounds could become valuable partners for use in surface plasmon resonance (SPR) biosensors in a photo-SPR modality. Acknowledgments We thank the US National Institutes of Health (Grants NS29632 and GM44836 to G.D.P.) for support and The University of Utah for providing funds for new initiatives. Special thanks are due to the Prestwich group co-workers whose published results are cited herein and to two conscientious reviewers for suggesting improvements to the text. References 1 Kotzyba-Hibert, F. et al. (1995) Recent trends in photoaffinity labeling. Angew. Chem., Int. Ed. Engl. 34, 1296–1312 2 Fedan, J. et al. (1984) Photoaffinity labels as pharmacological tools. Biochem. Pharmacol. 33, 1167–1180

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REVIEWS 28 Radding, J.A. et al. (1998) Photoaffinity analog of the semisynthetic echinocandin LY303366: identification of echinocandin targets in Candida albicans. Antimicrob. Agents Chemother. 42, 1187–1194 29 Bömmel, H.M. et al. (1998) Anti-pterins as tools to characterize the function of tetrahydrobiopterin in NO synthase. J. Biol. Chem. 273, 33142–33149 30 Abe, I. et al. (1998) Photoaffinity labeling of oxidosqualene cyclase and squalene cyclase by a benzophenone-containing inhibitor. Biochemistry 37, 5779–5784 31 Dang, T. et al. (1999) The binding site for an inhibitor of squalene:hopene cyclase determined using photoaffinity labeling and molecular modeling. Chem. Biol. 6, 333–341 32 Taylor, M.F. et al. (1996) Photoaffinity labeling of rat steroid 5 alphareductase (isozyme-1) by a benzophenone derivative of a 4-methyl4-azasteroid. Steroids 61, 323–331 33 Jamil, H. et al. (1998) Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells. J. Lipid Res. 39, 1448–1454 34 Otto, J. and Smith, W. (1996) Photolabeling of prostaglandin endoperoxidase H synthase-1 with 3-trifluoro-3-(m[125I]iodophenyl diazirine as a probe of membrane association and the cyclooxygenase active site. J. Biol. Chem. 271, 9906–9910 35 Mourey, R.J. et al. (1993) Inositol 1,4,5-trisphosphate receptors: mapping the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands. Biochemistry 32, 1719–1726 36 Chaudhary, A. et al. (1998) Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I. Chem. Biol. 5, 273–281 37 Tall, E. et al. (1997) Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs. Biochemistry 36, 7239–7248 38 Prestwich, G.D. et al. (1999) Probing phosphoinositide polyphosphate binding to proteins. In Phosphoinositides: Chemistry, Biochemistry and Biomedical Applications (Vol. 818) (Bruzik, K.S., ed.), pp. 24–37, American Chemical Society 39 Prestwich, G.D. (1996) Touching all the bases: inositol polyphosphate and phosphoinositide affinity probes from glucose. Acc. Chem. Res. 29, 503–513 40 Profit, A.A. et al. (1998) Probing the phosphoinositide binding site of the clathrin assembly protein AP-2 with photoaffinity labels. Arch. Biochem. Biophys. 357, 85–94 41 Chaudhary, A. et al. (1998) Specific interaction of Golgi coatomer a-COP with phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273, 8344–8350 42 Beck, W. and Qian, X. (1992) Photoaffinity substrates for P-glycoprotein. Biochem. Pharmacol. 43, 89–93 43 Boer, R. et al. (1996) Interaction of cytostatics and chemosensitizers with the dexniguldipine binding site on p-glycoprotein. Eur. J. Pharmacol. 295, 253–260 44 Wu, Q. et al. (1998) Identification of the domains of photoincorporation of the 39 and 7-benzophenone analogues of taxol in the carboxyl-terminal half of murine mdr1b P-glycoprotein. Biochemistry 37, 11272–11279 45 Ojima, I. et al. (1995) A new paclitaxel photoaffinity analog with a 3-(4-benzoylphenyl)propanoyl probe for characterization of drugbinding sites on tubulin and p-glycoprotein. J. Med. Chem. 38, 3891–3894 46 Chen, G.P. et al. (1997) Photoimmobilization of sulfated hyaluronic acid for antithrombogenicity. Bioconjugate Chem. 8, 730–734 47 Herbert, C.B. et al. (1997) Micropatterning gradients and controlling surface densities of photoactivatable biomolecules on self-assembled monolayers of oligo(ethylene glycol) alkanethiolates. Chem. Biol. 4, 731–737 48 Chevelot, Y. et al. (1999) Synthesis and characterization of a photoactivatable glycoaryldiazirine for surface glycoengineering. Bioconjugate Chem. 10, 169–175 49 Hypolite, C. et al. (1997) Formation of microscale gradients of protein using heterobifunctional photolinkers. Bioconjugate Chem. 8, 658–663 50 Ayadim, M. and Soumillion, J. (1995) Photoimmobilization covalently anchored to the silica surface: modulation of the excitation state efficiency through electron transfer from the linking arm or from the surface. Tetrahedron Lett. 36, 4615–4618

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51 Willner, I. and Rubin, S. (1996) Control of the structure and functions of biomaterials by light. Angew. Chem., Int. Ed. Engl. 35, 367–385 52 Niu, L. et al. (1996) Synthesis and photochemical properties of a kainate precursor and activation of kainate and AMPA receptor channels on a microsecond time scale. Biochemistry 35, 2030–2036 53 Rossi, F. et al. (1997) N-Nmoc-L-glutamate, a new caged glutamate with high chemical stability and low prephotolysis activity. J. Biol. Chem. 272, 32933–32939 54 Gee, K. et al. (1996) Desyl esters of amino acid neurotransmitters. Phototriggers for biologically active neurotransmitters. J. Org. Chem. 61, 1228–1233 55 Peng, L. and Goeldner, M. (1996) Synthesis and characterization of photolabile choline precursors as reversible inhibitors of cholinesterases: release of choline in the microsecond time range. J. Org. Chem. 61, 185–191 56 Furuta, T. et al. (1999) Brominated 7-hydroxycoumarin-4ylmethyls: photolabile protecting groups with biologically useful cross-sections for two photon photolysis. Proc. Natl. Acad. Sci. U. S. A. 96, 1193–1200 57 Finch, E. and Augustine, G. (1999) Local calcium signaling by inositol-1,4,5-trisphosphate in Purkinje cell dendritres. Nature 396, 753–756 58 Li, W.H. et al. (1997) Membrane-permeant esters of inositol polyphosphates, chemical syntheses and biological applications. Tetrahedron 53, 12017–12040 59 Jiang, T. et al. (1998) Membrane-permeant esters of phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273, 11017–11024 60 Qiao, L. et al. (1998) Synthesis and evaluation of a photolyzable derivative of sphingosine 1-phosphate: caged SPP. Bioorg. Med. Chem. Lett. 8, 711–714 61 Shapiro, R. et al. (1997) Identification of a ganglioside recognition domain of tetanus toxin using a novel ganglioside photoaffinity ligand. J. Biol. Chem. 272, 30380–30386 62 Odaka, M. et al. (1996) Synthesis, photoreactivity, and cytotoxic activity of caged compounds of leucyl-leucine methyl ester, an apoptosis inducer. Photochem. Photobiol. 63, 800–806 63 Curley, K. and Lawrence, D. (1999) Light-activated proteins. Curr. Opin. Chem. Biol. 3, 84–88 64 Wood, J. et al. (1998) A caged protein kinase inhibitor. J. Am. Chem. Soc. 120, 7145–7146 65 Curley, K. and Lawrence, D.S. (1998) Photoactivation of a signal transduction pathway in living cells. J. Am. Chem. Soc. 120, 8573–8574 66 Pan, P. and Bayley, H. (1997) Caged cysteine and thiophosphoryl peptides. FEBS Lett. 405, 81–85 67 Chang, C. et al. (1998) Caged catalytic subunit of cAMP-dependent protein kinase. J. Am. Chem. Soc. 120, 7661–7662 68 Cornish, V. and Schultz, P. (1994) A new tool for studying protein structure and function. Curr. Opin. Struct. Biol. 4, 601–607 69 Short, G. et al. (1999) Caged HIV-1 protease: dimerization is independent of the ionization state of the active site aspartates. J. Am. Chem. Soc. 121, 478–479 70 Arroyo, J. et al. (1997) In vivo photoactivation of caged thrombin. Thromb. Haemost. 78, 791–793 71 Wei, Y. et al. (1998) A photoactivated prodrug. Bioorg. Med. Chem. Lett. 8, 2419–2422 72 Reinhard, R. and Schmidt, B. (1998) Nitrobenzyl-based photosensitive phosphoramide mustards: synthesis and photochemical properties of potential prodrugs for cancer therapy. J. Org. Chem. 63, 2434–2441 73 Hogenkamp, H.P.C. et al. (1999) The pharmacological uses of cobalamin bioconjugates. In B-12 (Vol. 2) (Banerjee, R., ed.), pp. 385–410, Wiley 74 Shiah, J. et al. (1998) Influence of pH on aggregation and photoproperties of N-(2-hydroxypropyl)-methacrylamide copolymer– meso-chlorine6 conjugates. Drug Dev. 5, 119–126 75 Ottl, J. et al. (1998) Preparation and photactivation of caged fluorophores and caged proteins using a new class of heterobifunctional, photocleavable cross-linking reagents. Bioconjugate Chem. 9, 143–151 76 Pirrung, M. and Huang, C. (1996) A general method for the spatially defined immobilization of biomolecules on glass surfaces using caged biotin. Bioconjugate Chem. 7, 317–321

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79 Nakatani, K. et al. (1998) p-Cyano substituted benzophenone as an excellent photophore for one-electron oxidation of DNA. Tetrahedron Lett. 39, 2779–2782 80 Fang, K. et al. (1998) A bifunctional photoaffinity probe for ligand/ receptor interaction studies. J. Am. Chem. Soc. 120, 8543–8544

Single-nucleotide polymorphism analysis by MALDI–TOF mass spectrometry Timothy J. Griffin and Lloyd M. Smith Single-nucleotide polymorphisms (SNPs) have great potential for use in genetic-mapping studies, which locate and characterize genes that are important in human disease and biological function. For SNPs to realize their full potential in genetic analysis, thousands of different SNP loci must be screened in a rapid, accurate and cost-effective manner. Matrix-assisted laser desorption-ionization–time-of-flight (MALDI–TOF) mass spectrometry is a promising tool for the high-throughput screening of SNPs, with future prospects for use in genetic analysis.

he Human Genome Project continues to produce sequence data, and it has become evident that there is substantial variation in the DNA sequence between two individuals at many points throughout the genome. Most commonly, sequence variation occurs at discrete, single-nucleotide positions referred to as single-nucleotide polymorphisms (SNPs), which are estimated to occur at a frequency of approximately one per 1000 nucleotides1–4. Therefore, for every 1000 nucleotides, the average nucleotide identity at one position will differ between any two copies of that chromosome at a substantial frequency throughout a population. SNPs are biallelic polymorphisms, that is, the nucleotide identity at these polymorphic positions is generally constrained to one of two possibilities in humans, rather than the four nucleotide possibilities that could occur, in principle4.

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or more SNPs has been proposed as an ultimate goal to enable effective genetic-mapping studies in large populations6. Therefore, technologies capable of genotyping thousands of SNP markers from large numbers of individual DNA samples in an accurate, rapid and cost-effective manner are needed to make these studies feasible. A wide variety of approaches to genotyping SNPs have been developed in recent years2,3,7,8; amongst the more promising technologies being developed is matrix-assisted laser desorption-ionization–time-offlight (MALDI–TOF) mass spectrometry (MS). This article gives a brief introduction to MALDI–TOF MS, an overview of approaches previously developed for SNP analysis, as well as recent advances in the field (Table 1) and an assessment of what the future holds for MALDI–TOF MS as a SNP-genotyping technology.

Single-nucleotide polymorphisms SNPs have important implications in human genetic studies. First, a subset of SNPs occurs within proteincoding sequences3,4. The presence of a specific SNP allele can be implicated as a causative factor in human genetic disorders. Therefore, screening for such an allele in an individual might enable the detection of a genetic predisposition to disease. Second, SNPs can be used as genetic markers for use in genetic-mapping studies2–5, which locate and identify genes of functional importance. It has been proposed that a set of 3000 biallelic SNP markers would be sufficient for wholegenome-mapping studies in humans; a map of 100 000

MALDI–TOF mass spectrometry MALDI was introduced in 1988 by Karas and Hillenkamp as a revolutionary method for ionizing and mass-analysing large biomolecules9. These investigators discovered that irradiation of crystals formed by suitable small organic molecules (called the matrix) with a short laser pulse at a wavelength close to a resonant absorption band of the matrix molecules caused an energy transfer and desorption process, producing gasphase matrix ions. More importantly, they found that if a low concentration of a non-absorbing analyte, such as a protein or nucleic acid molecule, was added to the matrix in solution and embedded in the solid matrix crystals formed by drying of the mixture, the nonabsorbing, intact analyte molecules were also desorbed into the gas-phase and ionized upon irradiation with the laser, facilitating their mass analysis. Typically, predominantly singly charged molecular ions (both negative and positive) are detected by MALDI–TOF

T.J. Griffin ([email protected]) is at the Department of Molecular Biotechnology, University of Washington, Box 357730, Seattle, WA, 98195-7730, USA. L.M. Smith ([email protected]) is at the Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI, 53706-1396, USA. TIBTECH FEBRUARY 2000 (Vol. 18)

0167-7799/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(99)01401-8

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