The handle
http://hdl.handle.net/1887/87273
holds various files of this Leiden University
dissertation.
Author: Lahav, D.
7
1
General Introduction
Glycosidases are important enzymes in the turnover of polysaccharides and glycoconjugates, and are involved in a range of human pathologies including genetic disorders such as Gaucher and Pompe disease, but also in various cancers.1,2 The discovery of potent and selective glycosidase inhibitors for fundamental glycobiology studies and as leads for drug discovery requires access to suitable (glycomimetic) compound libraries, as well as easily applicable assays and screening formats. The research described in this Thesis was aimed to explore how covalent and irreversible glycosidase inhibitors can be applied in the screening of focused compound libraries on various glycosidases using fluorescence polarization activity-based protein profiling (FluoPol-ABPP).
1.1 Detection methods within activity-based protein profiling
Activity-based protein profiling (ABPP) is a powerful method for determining the number of active copies of specific enzymes in a given biological sample and under physiological conditions.3 Protein expression levels do not always correlate to the amount of active enzyme molecules, which might be regulated by post-translational modifications, protein-protein interactions or endogenous small-molecule based inhibition. Measuring mRNA or protein levels could therefore lead to misinterpretation of true enzyme activity levels and maybe even to misinterpreting the physiological role of an enzyme (or enzyme family).
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enzyme-probe complex.6 The type of detection depends on the reporter group.
Probes containing a fluorophore can be detected after separation of the protein content of the treated proteome via gel-electrophoresis, as in SDS-PAGE, using fluorescent imaging (Figure 1A). Another commonly used procedure is to detect and identify ABP-modified enzymes by mass spectrometry (MS)-based proteomics. Protein that have reacted with biotinylated probes are enriched via streptavidin pull-down and then digested with trypsin, producing peptide fragments derived from the ABP labelled protein(s). These fragments are subsequently identified with MS/MS sequencing (Figure 1B). Combination-ABPs containing a fluorophore and a biotin exist as well, and bioorthogonal chemistry can be employed where direct labelling falls short.7
Figure 1. Schematic representation of a typical ABPP experiment where labelled enzymes are either
directly visualized with SDS-PAGE followed by fluorescent imaging (A) or analysed by mass spectrometry (B). When using FluoPol as readout (C), high polarization signals are obtained when a high fluorescent signal parallel to the plane of excitation is retrieved. Generally this occurs when the apparent size of the fluorophores is large and thus when molecular mobility is relatively low. Free probe (bottom situation) will lead to a depolarized signal due to increased molecular tumbling and scattering of fluorescent light.
General Introduction
9 (in part) formation of probe-enzyme complex, with no (or diminished) in-gel fluorescence as the result. In such a competitive ABPP experiment the probe binding, reflected by the intensity of in-gel fluorescence, is inversely proportional to the degree of competition that occurs, and thus inhibitory potency of the competing compound. Gel-ABPP experiments are time-consuming and labour intensive processes when examining large numbers of compounds. Fluorescence polarization (FluoPol) as readout to monitor probe binding to the target enzyme allows the development of high-throughput screening compatible assays within the ABPP methodology, allowing fast identification of inhibitors or active site competitors.
In FluoPol, excitation of the fluorophore occurs with linear polarized light. The orientation of emitted light and thereby the degree of polarized light parallel to the plane of excitation depends on the movement the excited fluorophore has undergone, which in turn depends on the size of the fluorophore. Large fluorescent moieties, such as those consisting of a fluorescent ABP covalently and irreversibly bound to a target enzyme, move and rotate slower compared to the (much smaller) unreacted and unbound fluorescent ABP. In competitive FluoPol ABPP (inclusion of putative inhibitors prior to exposure of the enzyme of interest to the fluorescent ABP) the degree of inhibition is inversely proportional to the FluoPol-signal. As FluoPol can be performed in microtiter plates, it is possible to screen large compound libraries efficiently for the identification of competitors that reduce or enhance enzyme labelling.
1.2 Targeting enzyme families via activity-based protein profiling
ABPs consist of a reactive group, normally an electrophile that may react with an enzyme active site nucleophile to form a covalent and irreversible bond.6 Probe specificity is determined by a recognition element (structural features resembling that of the substrate) and a reporter moiety completes the ABP design. The nature of the electrophile depends on that of the nucleophile – and in broader terms the reaction mechanism – of the enzyme (or enzyme family) subject of the projected ABPP study. Fluorophosphonates, such as diisopropyl fluorophosphonate and its derivatives irreversibly inhibit serine hydrolases according to the mechanism shown in Figure 2 (entry 1) and are used as class-wide ABPs for serine hydrolases.8 Active site cysteine thiols in lysosomal cathepsins may covalently and irreversibly react with peptide vinyl sulfones as well as peptide epoxysuccinates, as shown in entries
10
of the cysteine thiol following a sequence of events also employed by the cysteine-containing cathepsins shown in entry 2.10 These studies show that specific classes of enzymes can be targeted using specific electrophiles. Introducing electrophilic traps within substrate mimetics, such as biotinylated acyl phosphates (entry 5) of ATP or ADP described by Patricelli et al.,11 demonstrates that this strategy also holds true for kinases. The work described in this Thesis builds on the successful design of electrophilic traps for yet another class of enzymes: the retaining glycosidases.
Figure 2. Structure and working mechanism of serine hydrolase probes (1), cathepsin probes (2 and 3), probes for ubiquitin specific proteases (4) and kinases (5).
1.3 Development of cyclophellitol-based ABPs for retaining glycosidases
In 1990 Atsumi et al.12 discovered that a culture filtrate of the mushroom strain,
Phellinus sp. was able to potently inhibit almond retaining β-glucosidase activity. It
β-General Introduction
11 glucosidase inhibition, and that inhibition proceeded through the mechanism as depicted in Figure 3A.13 Hansen et al.14 described in 2005 an efficient synthesis of large quantities of this natural product, starting from D-xylose. Though not the first
synthesis of this natural product, the authors were able to produce useful quantities of the natural product following a route that appeared attractive for adaptation towards structural, configurational and functional cyclophellitol analogues.15 The synthetic material obtained in the Madsen studies was used to reveal that retaining β-glucosidase from Thermotoga maritima reacts in a mechanism-based, covalent and irreversible manner with cyclophellitol (6) to form a covalent adduct, with the reacted inhibitor adopting a 4C1 chair conformation, as
witnessed by crystal studies on the enzyme-inhibitor adduct (figure 3A).16
This binding mode, indeed this mode of reaction of cyclophellitol with retaining β-glucosidases can be understood by perusing the molecular pathway through which retaining β-glucosidases process their substrates. Hydrolysis of interglycosidic linkages by retaining glycosidases mostly occurs via a two-step, double displacement mechanism involving a covalent glycosyl-enzyme intermediate as was proposed by Koshland in 1953.17,18 For retaining β-glucosidases, in the first step, (the glycosylation step), the aglycon is protonated through the action of an acid-base residue (either a glutamic acid or aspartic acid depending on the nature of the retaining β-glucosidase) and expelled leaving an oxocarbenium-ion like intermediate. In this transition state, the oxocarbenium ion can adopt a 4H3
half-chair conformation, (see figure 3B) that is then trapped by the catalytic nucleophile (aspartate or glutamate) to form the covalent adduct in a 4C1 chair conformation. In
the second displacement reaction, the deglycosylation step, hydrolysis of the formed covalent intermediate also occurs via an oxocarbenium ion intermediate in
4
H3 conformation. The covalent glycosyl-enzyme intermediate formed in the
12
Figure 3. (A) Chemical structures in of (+)-cyclophellitol (6). Also the mechanism-based inhibition is
depicted. (B) Mechanism of hydrolysis of β-glucosidic linkages by retaining β-glucosidases.
General Introduction
13 following formation of a covalent enzyme-glycoside intermediate and in many cases are bona fide irreversible inhibitors – thus allowing in-depth structural studies. The initial binding and reaction rate is however also slow, much slower than that of cyclophellitol-based compounds. Thus, and though activity-based probes based on 2-deoxy-2-fluoroglycosides and related compounds have been put forward by the groups of Vocadlo and Bertozzi,22 these studies have not been followed up much and this may be because deoxy-fluoroglycosides are – as evident from the above – often relatively poor retaining glycosidase inhibitors.24 In contrast, ABPs based on cyclitol epoxides and especially cyclitol aziridines have been developed in recent years that target a rather wide range of retaining glycosidases. These include GH1 β-glucosidases25, GH27 α-galactosidases26 and GH29 α-fucosidases27, retaining glycosidases that can be addressed by cyclophellitol-aziridine ABPs featuring configuration and substitution pattern
emulating that of the underlying substrate glycosides.28
Figure 4. Chemical structures of cyclophellitol (7) and cyclophellitol aziridine (8)-based probes with
two typical reporter groups (Tags) depicted in the middle. On the right two deoxy-fluoro-glycosides, 9 and 10, based on which activity-based glycosidase probes can be developed as well.
1.4 Aim and outline of this thesis
The research described in this thesis focuses on the development of FluoPol-ABPP assays for the discovery of competitive inhibitors for a number of retaining glycosidases involved in human disease. In Chapter 2 the development of FluoPol-ABPP for the identification of inhibitors of the human cytosolic retaining β-glucosidase, GBA2 is described. These studies, apart from yielding known and new competitive GBA2 inhibitors, also paved the way for the development of related assays directed at other retaining glycosidases. Chapter 3 describes the identification, using a FluoPol-ABPP assay format, of GBA1 inhibitors from the Library of Pharmaceutically Active Compounds (LOPAC1280, Sigma). Several of these
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inhibitors that might be used as pharmacological chaperones (stabilizing endogenous lysosomal retaining α-glucosidase) for the treatment of Pompe disease. Chapter 5 describes the development of FluoPol-ABPP on Golgi α-mannosidase II (GMII) from drosophila, a homologue of human GMII, which is a pharmacological target for chemotherapeutic agents. In each chapter a screen of an in-house (Leiden Institute of Chemistry, Leiden University) amassed iminosugar library, containing 358 entries as shown in appendix A, is performed with the optimal FluoPol-ABPP conditions. Chapter 6 gives a summary of the results described in this Thesis and discusses some future prospects based on the presented results.
1.5 References
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General Introduction
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W. E., van den Nieuwendijk, A. M. C. H., Bleijlevens, B., Kramer, G., Florea, B. I., Hooibrink, B., Hollak, C. E. M., Ottenhoff, R., Boot, R. G., van der Marel, G. a, Overkleeft, H. S., and Aerts, J. M. F. G. (2010) Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat. Chem. Biol. 6, 907–913.
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(21) Withers, S. G., Rupitz, K., and Street, I. P. (1988) 2-Deoxy-2-fluoro-D-glycosyl
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