Lianne I. Willems

Triple Bioorthogonal Ligation Strategy for Simultaneous Labeling of Multiple Enzymatic Activities

Bioorthogonal chemistry plays an important role in chemical biology research by creating the means to carry out selective chemical transformations in complex biological samples. A ligation reaction classifies as being bioorthogonal when it can be performed in a biological sample in a chemoselective manner without any interference with the biological system. Bioorthogonal reactions have been used in cell-surface labeling of glycoproteins and studies of biological processes that involve post-translational modifications. Another area of research that has benefited from bioorthogonal chemistry is two-step activity-based protein profiling (ABPP), where it enables the temporal separation of a reporter group and a chemical probe that is directed to the active site of an enzyme (such a chemical probe is also called activity-based probe, ABP). Two-step ABPP strategies are of particular interest when the presence of a tag interferes with selectivity, affinity, cell-permeability, or bioavailability of the probe. A further advantage of tandem labeling strategies is the option to use different reporter groups depending on the type of experiment and the desired method of analysis while using a single ABP. Several bioorthogonal ligation strategies have been described, and continuing efforts are being made to develop ligations that are more selective and efficient than existing methods. At the same time the high complexity of biological processes often requires the study of multiple targets simultaneously, thereby creating a need for ligation reactions that are orthogonal with respect to each other and can thus be used concurrently in a single experiment. Over the past decade, a number of tandem ligation strategies has been described for use in bioconjugation. The first report of a tandem bioorthogonal ligation in complex biological samples involved a Staudinger–Bertozzi ligation and Diels– Alder cycloaddition procedure, which utilizes mutually orthogonal reagents but suffers from the need to mask free thiol groups prior to the ligation step to avoid nonspecific labeling. More recently, it was reported that a copper-free azide–cyclooctyne cycloaddition can be used concurrently with an inverse-electron-demand Diels–Alder reaction between tetrazine and trans-cyclooctene for the simultaneous labeling of two different receptors on cell surfaces, provided that the proper reagents are carefully selected so that crossreactivity is minimized. Herein, we describe a triple ligation strategy employing the tetrazine ligation, Staudinger–Bertozzi ligation, and copper(I)-catalyzed Huisgen [2+3] cycloaddition (“click” reaction) for the selective and simultaneous labeling of three different enzymatic activities in a single experiment (Scheme 1a). Several examples of two-step ABPP strategies using click chemistry and Staudinger–Bertozzi ligation have been described previously. The tetrazine ligation, however, has thus far not been used for this purpose. Therefore we set out to develop a two-step ABPP strategy in which an ABP is functionalized with norbornene as a ligation handle that can react with a tetrazine reagent conjugated to a reporter group to enable detection and analysis of labeled proteins. As a model system for our studies we selected the 20S proteasome, containing three catalytically active subunits (b1, b2, and b5) that can be targeted by either broad-spectrum or subunit-specific ABPs. We designed two proteasome ABPs that are functionalized with norbornene as a ligation handle: ABP 1 is derived from the pan-reactive proteasome inhibitor epoxomicin, and ABP 2 has a different scaffold based on a b5-subunit-selective proteasome inhibitor (Scheme 1 b). Furthermore, we chose to create a panel of three tetrazine reagents functionalized with different tags, being BodipyTMR (3 a), BodipyFL (3 b), and biotin (3c). Other reagents used herein for two-step labeling of the proteasome by click chemistry and Staudinger ligation are shown in Scheme 1c. The synthesis of all reagents and competition experiments confirming the ability of the ABPs to target all proteolytically active proteasome b subunits (1, 4, 5) or only the b5 subunit (2) in cell extracts and/or in living cells can be found in the Supporting Information. The applicability of the tetrazine ligation for two-step labeling of endogenous proteasome activity was tested by exposing human embryonic kidney (HEK) cell lysates to norbornene-functionalized ABP 1 in a concentration that results in complete proteasome binding followed by ligation with one of the tetrazine reagents 3a–c for one hour at 37 8C. Analysis of labeled proteins by SDS-PAGE using either fluorescent readout or detection by streptavidin Western blotting (Figure 1a and Figure S2 in the Supporting Information) showed that ligation with all three tetrazine reagents results in labeling of the three catalytically active proteasome b subunits in a concentration-dependent manner. In this [*] L. I. Willems, N. Li, Dr. B. I. Florea, M. Ruben, Prof. G. A. van der Marel, Prof. H. S. Overkleeft Leiden Institute of Chemistry and Netherlands Proteomics Centre Gorlaeus Laboratories Einsteinweg 55, 2333 CC Leiden (The Netherlands) E-mail: h.s.overkleeft@chem.leidenuniv.nl

Novel Activity‐Based Probes for Broad‐Spectrum Profiling of Retaining β‐Exoglucosidases In Situ and In Vivo

A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases (see picture). These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.

Current Developments in Activity-Based Protein Profiling

Activity-based protein profiling (ABPP) has emerged as a powerful strategy to study the activity of enzymes in complex proteomes. The aim of ABPP is to selectively visualize only the active forms of particular enzymes using chemical probes termed activity-based probes (ABPs). These probes are directed to the active site of a particular target protein (or protein family) where they react in a mechanism-based manner with an active site residue. This results in the selective labeling of only the catalytically active form of the enzyme, usually in a covalent manner. Besides the monitoring of a specific enzymatic activity, ABPP strategies have also been used to identify and characterize (unknown) protein functions, to study up- and down-regulation of enzymatic activity in various disease states, to discover and evaluate putative new enzyme inhibitors, and to identify the protein targets of covalently binding natural products. In this Topical Review we will provide a brief overview of some of the recent developments in the field of ABPP.

Gaucher disease and Fabry disease: New markers and insights in pathophysiology for two distinct glycosphingolipidoses

Gaucher disease (GD) and Fabry disease (FD) are two relatively common inherited glycosphingolipidoses caused by deficiencies in the lysosomal glycosidases glucocerebrosidase and alpha-galactosidase A, respectively. For both diseases enzyme supplementation is presently used as therapy. Cells and tissues of GD and FD patients are uniformly deficient in enzyme activity, but the two diseases markedly differ in cell types showing lysosomal accumulation of the glycosphingolipid substrates glucosylceramide and globotriaosylceramide, respectively. The clinical manifestation of Gaucher disease and Fabry disease is consequently entirely different and the response to enzyme therapy is only impressive in the case of GD patients. This review compares both glycosphingolipid storage disorders with respect to similarities and differences. Presented is an update on insights regarding pathophysiological mechanisms as well as recently available biochemical markers and diagnostic tools for both disorders. Special attention is paid to sphingoid bases of the primary storage lipids in both diseases. The value of elevated glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease for diagnosis and monitoring of disease is discussed as well as the possible contribution of the sphingoid bases to (patho)physiology. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Proteasome Activity Imaging and Profiling Characterizes Bacterial Effector Syringolin A

Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: β1, β2, and β5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets β2 and β5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to β2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.

Synthesis of pH-activatable red fluorescent BODIPY dyes with distinct functionalities.

A series of tunable pH-dependent BODIPY dyes were synthesized and further functionalized in a Knoevenagel condensation reaction with various aldehydes. In this fashion, monofunctional dyes containing an alkyne, azide, or carboxylic acid (masked as its methyl ester) as ligation sites as well as asymmetrical bifunctional dyes were obtained, without compromising their pH-dependency. In addition, fluorescence excitation and emission maxima for these dyes were shown to be significantly red-shifted in comparison to their tetramethyl precursors.

A panel of subunit-selective activity-based proteasome probes

Mammals express seven different catalytically active proteasome subunits. In order to determine the roles of the different proteolytically active subunits in antigen presentation and other cellular processes, highly specific inhibitors and activity-based probes that selectively target specific active sites are needed. In this work we present the development of fluorescent activity-based probes that selectively target the beta1 and beta5 sites of the constitutive proteasome.

Azido-BODIPY Acid Reveals Quantitative Staudinger–Bertozzi Ligation in Two-Step Activity-Based Proteasome Profiling

Activity-based protein profiling (ABPP) research is directed towards the development of tools and techniques that report on enzyme activity in complex biological samples.[1–4] With the aid of activity-based probes (ABPs)—small molecules designed to react specifically, covalently, and irreversibly with the active site residues of an enzyme or enzyme family—enzymatic activity levels are detected, rather than the protein expression levels that are measured by means of conventional proteomics techniques. A typical ABP consists of three parts: 1) a “warhead”, the reactive group that binds covalently and irreversibly to the enzyme active site, 2) a recognition element targeting the ABP to a certain enzyme (family), and 3) an affinity tag or a fluorophore for visualization and/or enrichment purposes. In most ABPs that report on enzyme activity, the reporter group is directly attached to the probe, with obvious advantages with respect to experimental design. Incorporation of, for instance, a biotin or large fluorophore in an ABP, however, might have a detrimental effect either on bioavailability (cell permeability) or on enzyme reactivity of the probe, or on both. With the aim of alleviating these problems, the two-step labeling approach is an important alternative in ABPP. We and Cravatt and co-workers simultaneously reported that this approach is also versatile in the profiling of enzyme families: namely the proteasome and serine hydrolases, respectively.[5, 6] In two-step ABPP approaches a small biocompatible reactive group, normally an azide or an acetylene, is introduced into an ABP. After covalent modification of a target protein (family), a reporter group is introduced in a chemoselective manner, by means either of Staudinger–Bertozzi ligation[6–8] or of Huisgen [2+3] cycloaddition (the “click reaction”, of which both copper(I)-catalyzed[5, 9–13] and copper-free[14,15] versions exist). Key to the success of such two-step ABPP experiments are the selectivity (in terms of cross-reactivity towards endogenous functional groups in a biological sample) and efficiency (in terms of chemical yields with which the azide- or acetylene-modified proteins are converted) of the chemoselective ligation step by which the reporter group is attached to the modified proteins. There are several reports on the selectivity of both Staudinger–Bertozzi and click ligations.[11,14] Here we describe a compatible set of one-step and two-step proteasome ABPs 4 and 6 (Scheme 1) and demonstrate that with these the efficiency of the Staudinger–Bertozzi ligation in the two-step ABPP of the proteasome catalytic activities is estimated to proceed in a quantitative fashion. Scheme 1 Reagents and conditions: a) N-hydroxysuccinimide, EDC, DCM, 2 h, 68 %. b) DBU, DMF, 5 min. c) HOBt, 1 min. d) 2, DiPEA, 30 min, 86 %. e) 5, 10 mol % CuSO4, 20 mol % sodium ascorbate, tBuOH/H2O 1:1, RT, 15 h, quant. The design of probes 4 and 6 is based on the new bifunctional azido-BODIPY acid derivative 1, which can be incorporated into ABPs and subsequently functionalized either before or after enzyme labeling by both Staudinger–Bertozzi and click ligation. We have recently demonstrated that the BODIPY-TMR-modified proteasome inhibitor 8 (MV151) labels all proteasome catalytic sites both in cell lysates and in living cells.[16] The capability to introduce a biotin moiety into 4 at will at any time in the profiling experiment provides flexibility in designing the optimal ABP (one-step or two-step), depending on the nature of the ABPP experiment. The title compound, azido-BODIPY acid 1, was synthesized by adaptation of the literature procedures for the synthesis of BODIPY-TMR[16,17] (Supporting Information) and was subsequently converted into the corresponding succinimidyl ester 2 (Scheme 1). Removal of the Fmoc protective group in the hexapeptide vinyl sulfone 3,[16] followed by condensation with azido-BODIPY-OSu 2, gave ABP 4. Copper(I)-catalyzed Huisgen [2+3] cycloaddition[9, 10] with biotin-propargylamide (5) gave rise to the fluorescent and affinity-tagged ABP 6. Having synthesized probes 4 and 6, we assessed their ability to label the proteolytically active proteasome subunits both in cell lysates (Figure 1) and in living cells (Figure 2). EL-4 cell lysates containing both the constitutive proteasome and the immunoproteasome[18] were treated with increasing concentrations of 4 or 6 for 1 h at 37°C. The lysates treated with 4 were then exposed to biotin-phosphane 7 for 1 h at 37°C. All samples were precipitated, and their protein contents were resolved by SDS-PAGE. Direct in-gel read-out of the wet gel slabs showed uniform labeling of the proteasome catalytic subunits (β1, β2, β5, β1i, β2i, β5i) by both ABPs in a concentration-dependent manner. The observed patterns are similar to those demonstrated previously (see the labeling pattern of 8, Figure 1A lane 10 for a representative example).[16] Preincubation with epoxomicin[19,20] (Figure 1A, lane 9, Figure 1C, lane 8) abolished all labeling, which further confirms the activity-based mechanism of ABPs 4 and 6. ABP 4 appears to be slightly more reactive than its biotinylated counterpart 6 (compare Figure 1A, lanes 3–5 and Figure 1C, lanes 3–5). Quantitative Staudinger–Bertozzi ligation on proteasome subunits modified by ABP 4 is evidenced by the gel shift of those samples exposed to 100 μM biotin-phosphane 7 (Figure 1A; compare lanes 3–7 and 8). The efficiency of the ligation is also apparent when the streptavidin blots we prepared from the same gels are compared (Figures 1B and D). Again, the two patterns are highly similar, and the intensities of the signals are similar for those experiments in which we applied 10 μM concentrations of either 4 or 6 (Figures 1B and D, lanes 7). Figure 1 Fluorescence readout (A and C) and streptavidin blot (B and D) of labeled proteasomes in cell lysate. A) and B) EL-4 cell lysates (25 μg total protein) were treated with 4 for 1 h at 37 °C, followed by Staudinger ligation (100 μM ... Figure 2 A) Fluorescence readout, and B) streptavidin blot of labeled proteasomes in living cells. Living EL4 cells were exposed to the indicated probes for 2 h at 37 °C, before being harvested and lysed. Lanes 3–6: 25 μg total protein ... The proteasome labeling potential of ABPs 4 and 6 in living cells was established by incubating EL-4 cells with either of the two probes at various concentrations for 2 h at 37°C. The exposed cells were harvested, washed, and lysed, and the lysates were processed as before (Figure 2). The outcomes of these experiments are highly reminiscent of those involving the ABPP labeling of lysates depicted in Figure 1. However, the main, and important, difference is found in the divergent labeling efficiency now observed for the two probes. In contrast with the proteasome profiling experiments on lysates, in which both probes appeared about equally efficient, we estimate that the two-step ABP 4 is at least five times more efficient in targeting the proteasome catalytic activities in living cells. As both probes are equally efficient in labeling proteasomes in lysates, this difference must be based on the relative cell permeabilities of the two probes. In conclusion, we have demonstrated the versatility of the bifunctional fluorophore azido-BODIPY acid 1 as a new tool in ABPP experiments. We have established that the Staudinger–Bertozzi ligation proceeds in quantitative yield under the conditions applied here. This result essentially means that two-step ABPP may proceed with an efficiency equal to that of contemporary one-step ABPP approaches with respect to protein tagging. The efficiency thus depends on the reactivity of the ABP towards the target protein (family), and not on the chemoselective ligation employed in the second step. The advantage of two-step ABPP is evident from the results presented here demonstrating that ABP 4 is better than biotinylated analogue 6 at labeling proteasomes in living cells. We expect that BODIPY derivative 1 will be useful to the chemical biology community outside the proteasome field for several reasons. Firstly, the system presented here should be of assistance in optimizing Staudinger–Bertozzi ligation conditions, in reaction time and in the amount of phosphane used with respect to the azido modified biomolecule, for instance. Further, azido-BODIPY acid 1 can be readily transposed to different ABPP experimental settings. These include not only those directed towards the profiling of different enzyme families (entailing the incorporation of 1 into other ABPs), but also those directed towards the development or employment of other bio-orthogonal ligation strategies. An obvious extension of the work reported here is evaluation of the efficiency of the Huisgen cycloaddition reaction, but modification of the azide in 1 to encompass reaction partners for new bio-orthogonal ligations are envisaged as well. We are currently pursuing research in these directions.

Small molecule antagonists for chemokine CCR3 receptors

The chemokine receptor CCR3 is believed to play a role in the development of allergic diseases such as asthma, atopic dermatitis, and allergic rhinitis. Despite the conflicting results that have been reported regarding the importance of eosinophils and CCR3 in allergic inflammation, inhibition of this receptor with small molecule antagonists is thought to provide a valuable approach for the treatment of these diseases. This review describes the structure–activity relationships (SAR) of small molecule CCR3 antagonists as reported in the scientific and patent literature. Various chemical classes of small molecule CCR3 antagonists have been described so far, including (bi)piperidine and piperazine derivatives, N‐arylalkylpiperidine urea derivatives and (N‐ureidoalkyl)benzylpiperidines, phenylalanine derivatives, morpholinyl derivatives, pyrrolidinohydroquinazolines, arylsulfonamides, amino‐alkyl amides, imidazole‐ and pyrimidine‐based antagonists, and bicyclic diamines. The (N‐ureidoalkyl)benzylpiperidines are the best studied class in view of their generally high affinity and antagonizing potential. For many of these antagonists subnanomolar IC50 values were reported for binding to CCR3 along with the ability to effectively inhibit intracellular calcium mobilization and eosinophil chemotaxis induced by CCR3 agonist ligands in vitro.

Acylazetine as a Dienophile in Bioorthogonal Inverse Electron-Demand Diels–Alder Ligation

A new bioorthogonal N-acylazetine tag, suitable for tetrazine mediated inverse electron-demand Diels-Alder conjugation, is developed. The tag is small and achiral. We demonstrate the usefulness of N-acylazetine-tetrazine based bioorthogonal chemistry in two-step activity-based protein profiling. The performance of the new tetrazinophile in the labeling of catalytically active proteasome subunits was comparable to that of the more sterically demanding norbornene tag.